Gdf15 fusion proteins and uses thereof

ABSTRACT

Fusion proteins containing a half-life extension protein, a linker, and a GDF15 protein are described. Also described are nucleic acids encoding the fusion proteins, recombinant cells thereof, compositions comprising the fusion proteins, and methods of using the fusion proteins for treating or preventing metabolic diseases, disorders or conditions.

CROSS REFERENCES TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 16/412,819, filed May 15, 2019, which is a divisional of U.S.patent application Ser. No. 15/586,463, filed on May 4, 2017, now issuedU.S. Pat. No. 10,336,812, which claims priority to U.S. ProvisionalPatent Application No. 62/333,886, filed on May 10, 2016. Eachdisclosure is incorporated herein by reference in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 200310 401C1 SEQUENCE LISTING.txt. The text fileis 403 KB, was created on Nov. 17, 2021, and is being submittedelectronically via Patent Center.

FIELD OF THE INVENTION

The invention relates to GDF15 fusion proteins. In particular, theinvention relates to a fusion protein comprising a half-life extensionprotein, a linker and a GDF15 protein, nucleic acids and expressionvectors encoding the fusion proteins, recombinant cells thereof, andpharmaceutical compositions comprising the fusion proteins. Methods ofproducing the fusion proteins and using the fusion proteins to treatmetabolic disorders are also provided.

BACKGROUND OF THE INVENTION

GDF15, a member of the TGFI3 family, is a secreted protein thatcirculates in plasma as a 25 kDa homodimer. Plasma levels of GDF15 rangebetween 150 and 1150 pg/ml in most individuals (Tsai et al., J CachexiaSarcopenia Muscle. 2012, 3: 239-243). Plasma levels of GDF15 areincreased under conditions of injury, cardiovascular disease and certaintypes of cancer. This upregulation is thought to be a cytoprotectivemechanism. High plasma levels of GDF15 are associated with weight lossdue to anorexia and cachexia in cancer, and in renal and heart failure.In a clinical trial, GDF15 levels were an independent predictor ofinsulin resistance in obese, non-diabetic subjects (Kempf et al., Eur.J. Endo. 2012, 167: 671-678). A study in twins showed that thedifferences in levels of GDF15 within twin pairs correlated to thedifferences in BMI within that pair, suggesting that GDF15 serves as along-term regulator of energy homeostasis (Tsai et al., PLoS One.2015,10(7):e0133362).

While GDF15 has been extensively studied as a biomarker for severalcardiovascular and other disease states, a protective role for GDF15 hasalso been described in myocardial hypertrophy and ischemic injury(Collinson, Curr. Opin. Cardiol. 2014, 29: 366-371; Kempf et al., Nat.Med. 2011, 17: 581-589; Xu et al., Circ Res. 2006, 98:342-50). GDF15 wasshown to play an important role in protection from renal tubular andinterstitial damage in mouse models of type 1 and type 2 diabetes(Mazagova et al., Am. J. Physiol. Renal Physiol. 2013; 305:F1249-F1264). GDF15 is proposed to have a protective effect againstage-related sensory and motor neuron loss, and it improves recoveryconsequent to peripheral nerve damage (Strelau et al., Neurosci. 2009,29: 13640-13648; Mensching et al., Cell Tissue Res. 2012, 350: 225-238).In fact, GDF15 transgenic mice were shown to have a longer lifespan thantheir littermate controls, which can indicate that this molecule servesas a long-term survival factor (Wang et al., Aging. 2014, 6: 690-700).

Numerous reports have demonstrated the improvement of glucose toleranceand insulin sensitivity in mouse models upon treatment with GDF15protein. Two independent strains of transgenic mice overexpressing GDF15have decreased body weight and fat mass, as well as improved glucosetolerance (Johnen et al., Nat. Med. 2007, 13:1333-1340; Macia et al.,PLoS One. 2012, 7:e34868; Chrysovergis et al., Int. J. Obesity. 2014,38: 1555-1564). Increases in whole-body energy expenditure and oxidativemetabolism were reported in GDF15 transgenic mice (Chrysovergis et al.,2014, Id.). These were accompanied by an increase in thermogenic geneexpression in brown adipose tissue and an increase in lipolytic geneexpression in white adipose tissue. Mice lacking the GDF15 gene haveincreased body weight and fat mass (Tsai et al., PLoS One. 2013,8(2):e55174). An Fc-fusion of GDF15 was shown to decrease body weightand improve glucose tolerance as well as insulin sensitivity in an obesecynomolgus monkey model when administered weekly over a period of sixweeks (WO 2013/113008).

The effects of GDF15 on body weight are thought to be mediated via thereduction of food intake and increased energy expenditure. GDF15 mayimprove glycemic control via body weight-dependent and independentmechanisms.

Together, these observations suggest that increasing levels of GDF15 canbe beneficial as a therapy for metabolic diseases. There is a need inthe art for GDF15-based compositions that can be used to treat orprevent metabolic diseases, disorders, or conditions.

BRIEF SUMMARY OF THE INVENTION

The invention satisfies this need by providing fusion proteins of GDF15that demonstrate increased solubility/stability and exhibit featuresthat indicate they can be used to treat or prevent metabolic diseases,disorders, or conditions. Such features include, for example, decreasedbody weight, increased glucose tolerance, and improved insulinsensitivity of a subject administered with a fusion protein according toan embodiment of the invention.

In one general aspect, the invention relates to a fusion proteincomprising: (a) a half-life extension protein, (b) a linker, and (c) aGDF15 protein, wherein the fusion protein is arranged from N-terminus toC-terminus in the order (a)-(b)-(c).

In an embodiment of the invention, the GDF15 protein is a human GDF15protein or a functional variant thereof In particular embodiments, theGDF15 protein comprises a mature GDF15 protein or functional variantthereof. In more particular embodiments, the GDF15 protein comprises anamino acid sequence having at least 90% sequence identity to SEQ ID NO:6-11. In other particular embodiments, the GDF15 protein comprises theamino acid sequence of SEQ ID NO:11, such as an amino acid sequenceselected from the group consisting of SEQ ID NOs: 6-11.

In an embodiment of the invention, the half life extension protein ishuman serum albumin (HSA) or a functional variant thereof. In particularembodiments, the half life extension protein comprises an amino acidsequence having at least 90% identity to SEQ ID NO: 1. In otherparticular embodiments, the half life extension protein comprises anamino acid sequence selected from the group consisting of SEQ ID NOs:1-3.

In an embodiment of the invention, the linker is a flexible linker. Inparticular embodiments, the flexible linker contains the sequence(GGGGS)n wherein n is 2 to 20, preferably 4 to 10 (SEQ ID NO: 129). Inanother embodiment of the invention, the linker of the fusion protein isa structured linker. In particular embodiments, the structured linkercontains the sequence (AP)n (SEQ ID NO: 144) or (EAAAK)n (SEQ ID NO:130), wherein n is 2 to 20, preferably 4 to 10.

In embodiments of the invention, the fusion protein comprises an aminoacid sequence having at least 90% sequence identity to SEQ ID NOs: 5,25-30, 36-37, 40, 48, 55-60 or 64-75. In particular embodiments of theinvention, the fusion protein comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 5, 25-30, 40, 55-60, and 70. Inmore particular embodiment of the invention, the fusion proteincomprises the amino acid sequence of SEQ ID NO: 60.

In another general aspect, the invention relates to an isolated nucleicacid molecule encoding a fusion protein of the invention.

In another general aspect, the invention relates to an expression vectorcomprising a nucleic acid molecule encoding a fusion protein of theinvention.

In another general aspect, the invention relates to a recombinant hostcell comprising a nucleic acid molecule encoding a fusion protein of theinvention.

In another general aspect, the invention relates to a method ofobtaining a fusion protein of the invention. The method comprises: (1)culturing a host cell comprising a nucleic acid molecule encoding thefusion protein under a condition that the fusion protein is produced,and (2) recovering the fusion protein produced by the host cell.

In another general aspect, the invention relates to a pharmaceuticalcomposition comprising a fusion protein of the invention and apharmaceutically acceptable carrier.

In another general aspect, the invention relates to a pharmaceuticalcomposition comprising a nucleic acid molecule encoding a fusion proteinof the invention and a pharmaceutically acceptable carrier.

In another general aspect, the invention relates to a kit comprising apharmaceutical composition of the invention.

In another general aspect, the invention relates to a method of treatingor preventing a metabolic disease, disorder or condition, the methodcomprising administering to a subject in need thereof a therapeuticallyor prophylactically effective amount of a pharmaceutical composition ofthe invention. In a particular embodiment, the pharmaceuticalcomposition is administered to the subject subcutaneously orintravenously.

According to embodiments of the invention, a method of treating ametabolic disorder selected from the group consisting of type 2diabetes, elevated glucose levels, elevated insulin levels, obesity,dyslipidemia, diabetic nephropathy, myocardial ischemic injury,congestive heart failure, or rheumatoid arthritis, in a subject in needthereof, comprises administering to the subject a therapeuticallyeffective amount of a pharmaceutical composition comprising a fusionprotein comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 5, 25-30, 40, 55-60, and 70 and apharmaceutically acceptable carrier. In a particular embodiment, themethod comprises administering to the subject a therapeuticallyeffective amount of a pharmaceutical composition comprising a fusionprotein comprising the amino acid sequence of SEQ ID NO: 60 and apharmaceutically acceptable carrier.

Other aspects, features and advantages of the invention will be apparentfrom the following disclosure, including the detailed description of theinvention and its preferred embodiments and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofthe invention, will be better understood when read in conjunction withthe appended drawings. It should be understood that the invention is notlimited to the precise embodiments shown in the drawings.

In the drawings:

FIGS. 1A and 1B show the crystal structure of GDF15 (SEQ ID NO: 6),where the disulfide pairing of the first and second Cysteine residues(C1-C2) formed a loop at the N terminus of the protein, also shown isthe crystal structure of TGF433 (SEQ ID NO: 139) for comparison.

FIG. 2 shows the effects of subcutaneous administration of fusionproteins according to embodiments of the invention, e.g., fusionproteins FP1 (SEQ ID NO: 60) and 6xHis-FP1 (SEQ ID NO: 26 with a 6xHistag attached at the N-terminus), on food intake in C57BL/6 mice, thecumulative food intake at 24 hours post-administration is depicted.Values shown within the bars are % reduction compared to vehicle (PBS)group±SEM; N=8 animals per group for all groups, except N=9 in FP1 16nmol/kg group. *-p<0.05, as compared to vehicle; p values werecalculated using One-way ANOVA and Tukey's test for multiplecomparisons.

FIG. 3 shows the effects of subcutaneous administration of FP1 and6xHis-FP1 (SEQ ID NO: 26 with a 6xHis tag attached at the N-terminus) onfood intake in Sprague-Dawley rats, the cumulative food intake at 48hours post-administration is depicted. Values shown within the bars are% reduction compared to vehicle (PBS) group±SEM; N=8 animals per group.*-p<0.05, as compared to vehicle; p values were calculated using One-wayANOVA and Tukey's test for multiple comparisons.

FIG. 4 shows the change in body weight of diet induced obese (DIO) miceduring treatment with FP1. Arrows indicate time (days) of subcutaneousadministration post initial dose (Day 0); N=8 animals per group.*-p<0.05, for FP1 1 nmol/kg group as compared to vehicle; #-p<0.05, forFP1 10 nmol/kg group as compared to vehicle; p values were calculatedusing Two-way RM ANOVA and Tukey's test for multiple comparisons.

FIGS. 5A and 5B show the blood glucose levels in DIO mice during an oralglucose tolerance test (OGTT) after 14 days of dosing of FP1 every 3days (q3d), the levels are expressed as the area under the curve. N=8animals per group. *-p<0.05, for FP1 1 nmol/kg group as compared tovehicle; p values were calculated using One-way ANOVA and Tukey's testfor multiple comparisons.

FIG. 6 shows the fed blood glucose levels in DIO mice during treatmentwith FP1. N=8 animals per group. *-p<0.05, as compared to vehicle; pvalues were calculated using Two-way RM ANOVA and Tukey's test formultiple comparisons.

FIG. 7 shows the 4 hour fasting homeostatic model assessment of insulinresistance (HOMA-IR) in DIO mice after 14 days of treatment with FP1.N=8 animals per group. *-p<0.05, as compared to vehicle; p values werecalculated using One-way ANOVA and Tukey's test for multiplecomparisons.

FIG. 8 shows the change in body weight in ob/ob mice during treatmentwith FP1 every 3 days (qd3). Arrows indicate time (days) of subcutaneousadministration post initial dose (Day 0); N=9 animals per group.*-p<0.05, for FP1 10 nmol/kg group as compared to vehicle; #-p<0.05, forFP1 1 nmol/kg group as compared to vehicle; p values were calculatedusing Two-way RM ANOVA and Tukey's test for multiple comparisons.

FIG. 9 shows the blood glucose levels in ob/ob mice during treatmentwith FP1. Arrows indicate time (days) of subcutaneous administrationpost initial dose (Day 0); N=9 animals per group. *-p<0.05, for FP1 10nmol/kg group as compared to vehicle; #-p<0.05, for FP1 1 nmol/kg groupas compared to vehicle; p values were calculated using Two-way RM ANOVAand Tukey's test for multiple comparisons.

FIG. 10 shows the mean (±standard deviation, SD) of the serum drugconcentration-time profile of FP1 following 2 mg/kg intravenous (IV) andsubcutaneous (SC) administration in C57BI/6 mice.

FIG. 11 shows the mean (±SD) of the serum drug concentration-timeprofile of FP1 following 2 mg/kg IV and SC administration inSprague-Dawley rats.

FIG. 12 shows the mean (±SD) of the serum drug concentration-timeprofile of FP1 following 1 mg/kg IV and SC administration in cynomolgusmonkeys, as determined by immunoassays.

FIG. 13 shows the serum concentration (ng/mL) of FP1 as an intact dimerover time following a single IV administration in cynomolgus monkeys, asdetermined by immuno-affinity (IA) capture-LCMS analysis.

FIG. 14 shows the serum concentration (ng/mL) of FP1 as an intact dimerover time following a single SC administration in cynomolgus monkeys, asdetermined by immuno-affinity capture-LCMS analysis.

FIG. 15 shows the concentration of FP1, represented as a % of thestarting concentration, after 0, 4, 24 and 48 hours of ex vivoincubation in plasma obtained from two human subjects (Sub), asdetermined by immunoassay.

FIG. 16 shows the average concentration of FP 1, represented as a % oftime 0, as an intact dimer after 0, 4, 24 and 48 hours of ex vivoincubation in plasma obtained from two human subjects (Sub), asdetermined by intact mass immuno-affinity capture-LCMS analysis.

FIG. 17 shows acute food intake in lean C57BL6N male mice before andafter the administration of various N-terminal deletion variants ofGDF15. (SEQ ID NOs: 92, 111, and 112, compared to wild type fusion withno deletion (SEQ ID 26 with a 6xHis tag attached at the N-terminus). N=8animals per group; *-p<0.05, as compared to vehicle; p values werecalculated using Two-way RM ANOVA and Tukey's test for multiplecomparisons.

FIG. 18 shows the effect of a single dose of FP2 on food intake inC57BL/6 mice; specifically, cumulative food intake at 24 hours postadministration is shown. Values shown within the bars are % reductioncompared to PBS group (mean+SEM); N=8 animals per group for all groups,except N=6 in 6xHis-FP1. **-p<0.01, ***-p<0.001, ****-p<0.0001; p valueswere calculated using Two-way ANOVA and Dunnetts's test for multiplecomparisons.

FIG. 19 shows cumulative food intake measured in Sprague-Dawley rats at24 hours post administration of a single dose of FP2. Values shownwithin the bars are % reduction compared to PBS group (mean+SEM); N=8animals per group. **-p<0.01, p values were calculated using Two-wayANOVA and Tukey's test for multiple comparisons.

FIG. 20 shows the percent change in body weight during treatment withFP2 q3d in DIO mice. Arrows indicate the time of subcutaneous injectionsof FP2; N=6 animals per group; *-p,0.05, as compared with the vehicle,using Two Way ANOVA and Tukey's test for multiple comparisons;

FIGS. 21A and 21B show the area under the curve (AUC) for the bloodglucose concentration levels during an OGTT test after 14 days of q3ddosing of FP2 in DIO mice. *-p<0.05, using One Way ANOVA and Tukey'smultiple comparisons test, using n=8 animals per group.

FIG. 22A shows the plasma insulin levels during an OGTT after 8 days ofq3d dosing of FP2 in DIO mice. *-p<0.05, Vehicle vs. FP2 (0.3 nmol/kg);FP2 (10 nmol/kg); and Rosiglitazone. #-p<0.05, as compared toRosiglitazone (10 mg/kg), using Two Way RM ANOVA and Tukey's multiplecomparisons test.

FIGS. 22B shows the AUC for the plasma insulin levels during an OGTTafter 8 days of q3d dosing of FP2 in DIO mice. *-<0.05, as compared toVehicle; #-p<0.05, as compared to Rosiglitazone.

FIG. 23 shows the fed blood glucose levels after 8 days of q3d dosing ofFP2 in DIO mice. *-p<0.05, as compared to Vehicle, using Two Way RMANOVA and Tukey's multiple comparisons test, n=8 animals per group.

FIG. 24 shows fasting HOMA-IR after 14 days of treatment with FP2 q3d,followed by 5-hour fast on Day 14, in DIO mice. *-p<0.05, as compared toVehicle, using One Way ANOVA and Tukey's multiple comparisons test, forn=8 animals per group.

FIG. 25 shows serum concentrations of FP2 following 2 mg/kg intravenous(IV) and 2 mg/kg subcutaneous (SC) administration in C57B1/6 mice.Values represent mean±SD (n=5 samples per timepoint).

FIG. 26 shows serum concentrations of FP2 following 2 mg/kg intravenous(IV) and 2 mg/kg subcutaneous (SC) administration in Sprague Dawleyrats. N=5 samples per time point.

FIG. 27 shows plasma concentrations of FP2 in cynomolgus monkeysanalyzed by immunoassay. Values represent mean±SD of n=3, except n=2 forIV at day 22 (528 hr). IV—intravenous, SC—subcutaneous.

FIG. 28 shows plasma concentrations of FP2 as intact dimer in cynomolgusmonkeys analyzed by LCMS. Values represent mean±SEM of n=3, except n=2for subcutaneous (SC) at 168 hours, n=1 for SC at 120 hours and 432hours, and n=1 for IV—intravenous at 168 hours and 432 hours.

FIG. 29 shows ex vivo stability of FP2 (Normalized Percent Recovery)over 48 hours in human plasma measured by immunoassay.

FIG. 30 shows ex vivo stability of FP2 (Normalized Percent Recovery)over 48 hours in human plasma measured by intact LC/MS.

FIG. 31 shows concentration response curves for FP2 and HSA-GDF15:GDF15heterodimer using pAKT Assays in SK-N-AS cells expressing recombinanthuman GFRAL receptor (N=3).

FIG. 32 shows daily food intake (g) prior to and following a single doseof FP1 in cynomolgus monkeys. *-p<0.05 for 10mg/kg of FP1 as compared tovehicle.

FIG. 33 shows percent body weight change prior to and following a singledose of FP1 in cynomolgus monkeys. *-p<0.05 for 10mg/kg FP1 as comparedto vehicle; #-p<0.05 for 3mg/kg as compared to vehicle using Two Way RMANOVA and Tukey's multiple comparisons test, for n=8 animals per group.

FIG. 34 shows daily food intake (g) prior to and following a single doseof FP2 in cynomolgus monkeys. *-p<0.05, as compared to Vehicle, usingTwo Way RM ANOVA and Tukey's multiple comparisons test, for n=8 animalsper group.

FIG. 35 shows percent body weight change prior to and following a singledose of FP2 in cynomolgus monkeys. *-p<0.05, for 10 nmol/kg of FP2 ascompared to Vehicle, #-p<0.05, for 3 nmol/kg of FP2 as compared toVehicle, & -p<0.05, for 1 nmol/kg of FP2 as compared to Vehicle, usingTwo Way RM ANOVA and Tukey's multiple comparisons test, for n=8 animalsper group.

DETAILED DESCRIPTION OF THE INVENTION

Various publications, articles and patents are cited or described in thebackground and throughout the specification; each of these references isherein incorporated by reference in its entirety. Discussion ofdocuments, acts, materials, devices, articles or the like which has beenincluded in the present specification is for the purpose of providingcontext for the invention. Such discussion is not an admission that anyor all of these matters form part of the prior art with respect to anyinventions disclosed or claimed.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning commonly understood to one of ordinary skill inthe art to which this invention pertains. Otherwise, certain terms usedherein have the meanings as set in the specification. All patents,published patent applications and publications cited herein areincorporated by reference as if set forth fully herein. It must be notedthat as used herein and in the appended claims, the singular forms “a,”“an,” and “the” include plural reference unless the context clearlydictates otherwise.

The invention relates to a fusion protein comprising (a) a halflife-extension protein, (b) a linker, and (c) a GDF15 protein, whereinthe fusion protein is arranged from N-terminus to C-terminus in theorder (a)-(b)-(c).

It is found that a fusion protein according to an embodiment of theinvention, comprising a half life-extension protein, a linker, and aGDF15 protein, results in an increased half life of the GDF15 protein,and fusion proteins of the invention exhibit metabolic effects thatdemonstrate their suitability as therapeutics for treating andpreventing metabolic diseases, disorders or conditions. Such effectsinclude, but are not limited to, decreasing body weight, increasingglucose tolerance, and improving insulin sensitivity of animalsadministered with the fusion proteins.

As used herein, the term “fusion protein” refers to a protein having twoor more portions covalently linked together, where each of the portionsis derived from different proteins.

Fusion proteins according to embodiments of the invention can includeany GDF15 protein. As used herein, the term “GDF15 protein” refers toany naturally-occurring wild-type growth differentiation factor 15protein or a functional variant thereof. The GDF15 protein can be fromany mammal, such as a human or another suitable mammal, such as a mouse,rabbit, rat, pig, dog, or a primate. In particular embodiments, theGDF15 protein is a human GDF15 protein or a functional variant thereof.In preferred embodiments, the GDF15 protein is a mature GDF15 protein ora functional variant thereof.

As used herein, the term “mature GDF15 protein” refers to the portion ofthe pre-pro-protein of GDF15 that is released from the full-lengthprotein following intracellular cleavage at the RXXR furin-like cleavagesite. Mature GDF15 proteins are secreted as homodimers linked bydisulfide bonds. In one embodiment of the invention, a mature GDF15protein, shorthand GDF15(197-308) (SEQ ID NO: 6), contains amino acids197-308 of a full-length human GDF15 protein.

As used herein, “functional variant” refers to a variant of a parentprotein having substantial or significant sequence identity to theparent protein and retains at least one of the biological activities ofthe parent protein. A functional variant of a parent protein can beprepared by means known in the art in view of the present disclosure. Afunctional variant can include one or more modifications to the aminoacid sequence of the parent protein. The modifications can change thephysico-chemical properties of the polypeptide, for example, byimproving the thermal stability of the polypeptide, altering thesubstrate specificity, changing the pH optimum, and the like. Themodifications can also alter the biological activities of the parentprotein, as long as they do not destroy or abolish all of the biologicalactivities of the parent protein.

According to embodiments of the invention, a functional variant of aparent protein comprises a substitution, preferably a conservative aminoacid substitution, to the parent protein that does not significantlyaffect the biological activity of the parent protein. Conservativesubstitutions include, but are not limited to, amino acid substitutionswithin the group of basic amino acids (arginine, lysine and histidine),acidic amino acids (glutamic acid and aspartic acid), polar amino acids(glutamine and asparagine), hydrophobic amino acids (leucine, isoleucineand valine), aromatic amino acids (phenylalanine, tryptophan andtyrosine), and small amino acids (glycine, alanine, serine, threonineand methionine). Non-standard or unnatural amino acids (such as4-hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid, isovaline,and alpha-methyl serine) can also be used to substitute standard aminoacid residues in a parent protein.

According to other embodiments of the invention, a functional variant ofa parent protein comprises a deletion and/or insertion of one or moreamino acids to the parent protein. For example, a functional variant ofa mature GDF15 protein can include a deletion and/or insertion of 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30 or more amino acids to the mature GDF15protein, preferably, a deletion of 1 to 30 amino acids at the N-terminusof the mature GDF15 protein.

According to yet other embodiments of the invention, a functionalvariant of a parent protein comprises a substitution, preferably aconservative amino acid substitution, and a deletion and/or insertion,preferably a small deletion and/or insertion of amino acids, to theparent protein.

According to embodiments of the invention, a fusion protein of theinvention comprises a GDF15 protein that has an amino acid sequence atleast 90% identical to the amino acid sequence of a mature GDF15, suchas GDF15(197-308) (SEQ ID NO: 6); or an amino acid sequence at least 90%identical to the amino acid sequence of a mature GDF15 truncated at theN-terminus, such as GDF15(200-308) (SEQ ID NO: 7), GDF15(201-308) (SEQID NO: 8), GDF15(202-308) (SEQ ID NO: 9), GDF15(203-308) (SEQ ID NO:10), or GDF15(211-308) (SEQ ID NO: 11). The GDF15 protein can have atleast one of substitutions, insertions and deletions to SEQ ID NO: 6, 7,8, 9, 10 or 11, as long as it maintains at least one of the biologicalactivities of the GDF15 protein, such as its effects on food intake,blood glucose levels, insulin resistance, and body weight, etc.

In particular embodiments, a fusion protein of the invention comprises aGDF15 protein having the amino acid sequence of SEQ ID NO: 11, includingbut not limited to, the amino acid sequence of SEQ ID NO: 6, 7, 8, 9, 10or 11.

Any suitable half life extension protein can be used in fusion proteinsaccording to embodiments of the invention. As used herein, the term“half life extension protein” can be any protein or fragment thereofthat is known to extend the half life of proteins to which it is fused.Examples of such half life extension proteins include, but are notlimited to, human serum albumin (HSA), the constant fragment domain (Fc)of an immunoglobulin (Ig), or transferrin (Tf). In embodiments of theinvention, the half life extension protein comprises HSA or a functionalvariant thereof. In particular embodiments of the invention, the halflife extension protein comprises an amino acid sequence that is at least90% identity to SEQ ID NO: 1. In preferred embodiments of the invention,the half life extension protein comprises HSA or functional variantthereof wherein the cysteine residue at position 34 of the HSA has beenreplaced by serine or alanine.

In particular embodiments, a fusion protein of the invention comprises ahalf life extension protein having an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 1-3.

Any suitable linker can be used in fusion proteins according toembodiments of the invention. As used herein, the term “linker” refersto a linking moiety comprising a peptide linker. Preferably, the linkerhelps insure correct folding, minimizes steric hindrance and does notinterfere significantly with the structure of each functional componentwithin the fusion protein. In some embodiments of the invention, thepeptide linker comprises 2 to 120 amino acids. For example, the peptidelinker comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105,106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119 or120 amino acids.

In embodiments of the invention, the linker increases the flexibility ofthe fusion protein components. In particular embodiments of theinvention, the linker can be a flexible linker comprising the sequence(GGGGS)n (SEQ ID NO: 129), including but not limited to, GS-(GGGGS)n SEQID NO: 142 or AS-(GGGGS)n-GT SEQ ID NO: 143, wherein n is 2 to 20, suchas 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

In other embodiments of the invention, the linker is structured. Inparticular embodiments of the invention, the linker can be a structuredlinker comprising the sequence (AP)n (SEQ ID NO: 144) or (EAAAK)n (SEQID NO: 130), including but not limited to, AS-(AP)n-GT (SEQ ID NO: 145)or AS-(EAAAK)n-GT (SEQ ID NO: 140), wherein n is 2 to 20, such as 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. In other embodiments of theinvention, the linker comprises the sequences (GGGGA)n (SEQ ID NO: 131),(PGGGS)n (SEQ ID NO: 132), (AGGGS)n (SEQ ID NO: 133) orGGS-(EGKSSGSGSESKST)n-GGS (SEQ ID NO: 134) wherein n is 2 to 20.

In embodiments of the invention, the fusion protein comprises an aminoacid sequence having at least 90% sequence identity to SEQ ID NOs: 5,25-30, 36-37, 40, 48, 55-56, 59-60 or 64-75. In particular embodimentsof the invention, the fusion protein comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 5, 25-30, 36-37, 40,48, 55-56, 59-60 and 64-75. In more particular embodiments of theinvention, the fusion protein comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 5, 25-30, 40, 55-56, 55-56,59-60, and 70. In further more particular embodiments of the invention,the fusion protein comprises the amino acid sequence of SEQ ID NO: 92,SEQ ID NO: 60 or SEQ ID NO: 26. The fusion protein can also includesmall extension(s) at the amino- or carboxyl-terminal end of theprotein, such as a tag that facilitates purification, such as apoly-histidine tag, an antigenic epitope or a binding domain.

The fusion proteins disclosed herein can be characterized or assessedfor GDF15 biological activities including, but not limited to effects onfood intake, oral glucose tolerance tests, measurements of blood glucoselevels, insulin resistance analysis, changes in body weight,pharmacokinetic analysis, toxicokinetic analysis, immunoassays and massspec analysis of the level and stability of full-length fusion proteins,and human plasma ex vivo stability analysis.

The invention also provides an isolated nucleic acid molecule encoding afusion protein of the invention. In embodiments of the invention, theisolated nucleic acid molecule encodes a fusion protein comprising anamino acid sequence having at least 90% sequence identity to SEQ ID NOs:5, 25-30, 36-37, 40, 48, 55-56, 59-60 or 64-75. In particularembodiments, the isolated nucleic acid molecule encodes a fusion proteincomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 5, 25-31, 36-37, 40, 48, 55-56, 59-60 and 64-75. In moreparticular embodiments, the isolated nucleic acid molecule encodes afusion protein comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 5, 25-30, 40, 55-56, 59-60, and 70. In furthermore particular embodiments, the isolated nucleic acid moleculecomprises the nucleotide sequence of SEQ ID NOs: 76-91.

According to other embodiments of the invention, the nucleic acidmolecule encoding the fusion protein can be in an expression vector.Expression vectors include, but are not limited to, vectors forrecombinant protein expression and vectors for delivery of nucleic acidsinto a subject for expression in a tissue of the subject, such as viralvectors. Examples of viral vectors suitable for use with the inventioninclude, but are not limited to adenoviral vectors, adeno-associatedvirus vectors, lentiviral vectors, etc. The vector can also be anon-viral vector. Examples of non-viral vectors include, but are notlimited to plasmids, bacterial artificial chromosomes, yeast artificialchromosomes, bacteriophages, etc. The vector can include any element toestablish a conventional function of an expression vector, for example,a promoter, ribosome binding element, terminator, enhancer, selectionmarker, or an origin of replication.

According to other embodiments of the invention, the nucleic acidmolecule encoding the fusion protein can be codon optimized for improvedrecombinant expression from a desired host cell, such as Human EmbryonicKidney (HEK) or Chinese hamster ovary (CHO) cells, using methods knownin the art in view of the present disclosure.

The invention also provides a host cell comprising a nucleic acidmolecule encoding a fusion protein of the invention. Host cells include,but are not limited to, host cells for recombinant protein expressionand host cells for delivery of the nucleic acid into a subject forexpression in a tissue of the subject. Examples of host cells suitablefor use with the invention include, but are not limited to HEK or CHOcells.

In another general aspect, the invention relates to a method ofobtaining a fusion protein of the invention. In a general aspect, themethod comprises: (1) culturing a host cell comprising a nucleic acidmolecule encoding a fusion protein under a condition that the fusionprotein is produced, and (2) recovering the fusion protein produced bythe host cell. The fusion protein can be purified further using methodsknown in the art.

In some embodiments, the fusion protein is expressed in host cells andpurified therefrom using a combination of one or more standardpurification techniques, including, but not limited to, affinitychromatography, size exclusion chromatography, ultrafiltration, anddialysis. Preferably, the fusion protein is purified to be free of anyproteases.

The invention also provides a pharmaceutical composition comprising afusion protein of the invention and a pharmaceutically acceptablecarrier.

The invention further provides a composition comprising a nucleic acidmolecule encoding a fusion protein of the invention and apharmaceutically acceptable carrier. Compositions comprising a nucleicacid molecule encoding a fusion protein of the invention can comprise adelivery vehicle for introduction of the nucleic acid molecule into acell for expression of the fusion protein. Examples of nucleic aciddelivery vehicles include liposomes, biocompatible polymers, includingnatural polymers and synthetic polymers, lipoproteins, polypeptides,polysaccharides, lipopolysaccharides, artificial viral envelopes, metalparticles, and bacteria, viruses, such as baculoviruses, adenovirusesand retroviruses, bacteriophages, cosmids, plasmids, fungal vectors andother recombination vehicles typically used in the art which have beendescribed for expression in a variety of eukaryotic hosts.

The pharmaceutically acceptable carrier can include one or more ofpharmaceutically acceptable excipient, buffer, stabilizer or othermaterials known to those skilled in the art. Examples ofpharmaceutically acceptable carriers include, but are not limited to,one or more of water, saline, buffer, isotonic agents such as sugars,polyalcohols, auxiliary substances such as wetting or emulsifyingagents, preservatives, as well as combinations thereof. Such materialsshould be non-toxic and should not interfere with the efficacy of theactive ingredient at the dosages and concentrations employed. Theprecise nature of the carrier or other material can depend on the routeof administration, e.g., intramuscular, subcutaneous, oral, intravenous,cutaneous, intramucosal (e.g., gut), intranasal or intraperitonealroutes. For example, liquid pharmaceutical compositions generallyinclude a liquid carrier such as water, petroleum, animal or vegetableoils, mineral oil or synthetic oil. Physiological saline solution,dextrose or other saccharide solution or glycols such as ethyleneglycol, propylene glycol or polyethylene glycol can also be included.Compositions for parenteral administration can be stored in lyophilizedform or in a solution, and are generally placed into a container havinga sterile access port, such as an intravenous solution bag or vialhaving a stopper pierceable by a hypodermic injection needle.

According to embodiments of the invention, a pharmaceutical compositioncan comprise one or more additional components, such as another activeingredient.

The invention also relates to kits comprising a pharmaceuticalcomposition of the invention. The kits can contain a first containerhaving a dried fusion protein of the invention and a second containerhaving an aqueous solution to be mixed with the dried fusion proteinprior to administration to a subject, or a single container containing aliquid pharmaceutical composition of the invention. The kit can containa single-dose administration unit or multiple dose administration unitsof a pharmaceutical composition of the invention. The kit can alsoinclude one or more pre-filled syringes (e.g., liquid syringes andlyosyringes). A kit can also comprise instructions for the use thereof.The instructions can describe the use and nature of the materialsprovided in the kit, and can be tailored to the precise metabolicdisorder being treated.

The invention also relates to use of the pharmaceutical compositionsdescribed herein to treat or prevent a metabolic disease, disorder orcondition, such as type 2 diabetes, elevated glucose levels, elevatedinsulin levels, obesity, dyslipidemia, diabetic nephropathy, myocardialischemic injury, congestive heart failure, or rheumatoid arthritis.According to embodiments of the invention, a method of treating orpreventing a metabolic disease, disorder or condition in a subject inneed of the treatment comprises administering to the subject atherapeutically or prophylactically effective amount of a pharmaceuticalcomposition of the invention. Any of the pharmaceutical compositionsdescribed herein can be used in a method of the invention, includingpharmaceutical compositions comprising a fusion protein of the inventionor pharmaceutical compositions comprising a nucleic acid encoding thefusion protein.

As used herein, “subject” means any animal, particularly a mammal, mostparticularly a human, who will be or has been treated by a methodaccording to an embodiment of the invention. The term “mammal” as usedherein, encompasses any mammal. Examples of mammals include, but are notlimited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits,guinea pigs, non-human primates (NHPs) such as monkeys or apes, humans,etc., more particularly a human.

A “metabolic disease, disorder or condition” refers to any disorderrelated to abnormal metabolism. Examples of metabolic diseases,disorders or conditions that can be treated according to a method of theinvention include, but are not limited to, type 2 diabetes, elevatedglucose levels, elevated insulin levels, obesity, dyslipidemia, diabeticnephropathy, myocardial ischemic injury, congestive heart failure, orrheumatoid arthritis.

The terms “treat,” “treating,” and “treatment” as used herein refer toadministering a composition to a subject to achieve a desiredtherapeutic or clinical outcome in the subject. In one embodiment, theterms “treat,” “treating,” and “treatment” refer to administering apharmaceutical composition of the invention to reduce, alleviate or slowthe progression or development of a metabolic disorder, such as type 2diabetes, elevated glucose levels, elevated insulin levels, obesity,dyslipidemia, diabetic nephropathy, myocardial ischemic injury,congestive heart failure, or rheumatoid arthritis.

The term “therapeutically effective amount” means an amount of atherapeutically active compound needed to elicit the desired biologicalor clinical effect. According to embodiments of the invention, “atherapeutically effective amount” is an amount sufficient to effectbeneficial or desired results, including clinical results. Atherapeutically effective amount can be administered in one or moreadministrations. In terms of a disease state, an effective amount is anamount sufficient to ameliorate, stabilize, or delay development of adisease. According to specific embodiments of the invention, atherapeutically effective amount is an amount of a fusion protein neededto treat or prevent a metabolic disease, disorder or condition, such astype 2 diabetes, elevated glucose levels, elevated insulin levels,obesity, dyslipidemia, diabetic nephropathy, myocardial ischemic injury,congestive heart failure, or rheumatoid arthritis.

According to embodiments of the invention, a pharmaceutical compositionof the invention can be administered to a subject by any method known tothose skilled in the art in view of the present disclosure, such as byintramuscular, subcutaneous, oral, intravenous, cutaneous, intramucosal(e.g., gut), intranasal or intraperitoneal route of administration. Inparticular embodiments, a pharmaceutical composition of the invention isadministered to a subject by intravenous injection or subcutaneousinjection.

Parameters such as the dosage amount, frequency of administration, andduration of administration of a pharmaceutical composition to a subjectaccording to an embodiment of the invention are not limited in anyparticular way. The optimum values of such parameters can depend on avariety of factors, such as the subject to be treated, the particularmetabolic disease to be treated, the severity of the disease, the routeof administration, etc., and one of ordinary skill in the art will beable to determine the optimum values for such parameters in order toachieve the desired therapeutic or clinical outcome. For example, apharmaceutical composition can be administered once per day, or morethan once per day, such as twice, three times, four times, etc. Atypical dosage can range from about 0.1 μg/kg to up to about 100 mg/kgor more of the fusion protein, depending on the factors such as thosementioned above.

EMBODIMENTS

Embodiment 1 is a fusion protein comprising: (a) a half-life extensionprotein, (b) a linker, and (c) a GDF15 protein; wherein the fusionprotein is arranged from N-terminus to C-terminus in the order(a)-(b)-(c).

Embodiment 2 is a fusion protein according to Embodiment 1, wherein theGDF15 protein is a human GDF15 protein or a functional variant thereof

Embodiment 3 is a fusion protein according to Embodiment 1, wherein theGDF15 protein comprises an amino acid sequence having at least 90%identity to an amino acid sequence selected from the group consisting ofSEQ ID NOs: 6-11.

Embodiment 4 is a fusion protein according to Embodiment 1, wherein theGDF15 protein comprises the amino acid sequence of SEQ ID NO: 11.

Embodiment 5 is a fusion protein according to Embodiment 4, wherein theGDF15 protein comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 6-11.

Embodiment 6 is a fusion protein according to any of Embodiments 1 to 5,wherein the half-life extension protein comprises human serum albumin(HSA) or a functional variant thereof.

Embodiment 7 is a fusion protein according to Embodiment 6, wherein thehalf-life extension protein comprises an amino acid sequence having atleast 90% identity to SEQ ID NO: 1.

Embodiment 8 is a fusion protein according to Embodiment 7, wherein thehalf-life extension protein comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 1-3.

Embodiment 9 is a fusion protein according to any of Embodiments 1 to 8,wherein the linker is a flexible linker.

Embodiment 10 is a fusion protein according to Embodiment 9, wherein thelinker comprises the sequence (GGGGS)n, wherein n is 2 to 20 (SEQ ID NO:129), such as GS-(GGGGS)x8 (SEQ ID NO: 12) or AS-(GGGGS)x8—GT (SEQ IDNO: 141).

Embodiment 11 is a fusion protein according to any of Embodiments 1 to9, wherein the linker is a structured linker.

Embodiment 12 is a fusion protein according to Embodiment 11, whereinthe linker comprises the sequence (AP)n (SEQ ID NO: 144) or (EAAAK)n(SEQ ID NO: 130), wherein n is 2 to 20, such as AS-(AP)n-GT (SEQ ID NO:145) or AS-(EAAAK)n-GT (SEQ ID NO: 140).

Embodiment 13 is a fusion protein comprising an amino acid sequencehaving at least 90% sequence identity to SEQ ID NOs: 5, 25-31, 36-37,40, 48, 55-60 or 64-75.

Embodiment 14 is a fusion protein according to Embodiment 13, comprisingan amino acid sequence selected from the group consisting of SEQ ID NO:5, 25-31, 36-37, 40, 48, 55-60 and 64-75.

Embodiment 15 is a fusion protein according to Embodiment 14, comprisingan amino acid sequence selected from the group consisting of SEQ ID NOs:5, 25-30, 40, 55-60, and 70.

Embodiment 16 is an isolated nucleic acid molecule encoding the fusionprotein of any one of Embodiments 1 to 15.

Embodiment 17 is an isolated nucleic acid molecule comprising thenucleotide sequence of SEQ ID NOs: 76-91.

Embodiment 18 is an expression vector comprising the nucleic acidmolecule of Embodiment 16 or 17.

Embodiment 19 is a host cell comprising the nucleic acid molecule ofEmbodiment 16 or 17.

Embodiment 20 is a method of producing the fusion protein of any one ofEmbodiments 1 to 15, comprising: (1) culturing a host cell comprising anucleic acid molecule encoding the fusion protein under a condition thatthe fusion protein is produced; and (2) recovering the fusion proteinproduced by the host cell.

Embodiment 21 is a method according to Embodiment 20, wherein therecovering step comprises purifying the fusion protein to removeproteases.

Embodiment 22 is a pharmaceutical composition comprising atherapeutically effective amount of the fusion protein of any one ofEmbodiments 1 to 15 and a pharmaceutically acceptable carrier.

Embodiment 23 is a pharmaceutical composition comprising atherapeutically effective amount of a nucleic acid molecule encoding thefusion protein of any one of Embodiments 1 to 15 and a pharmaceuticallyacceptable carrier.

Embodiment 24 is a kit comprising a pharmaceutical composition accordingto Embodiment 22 or 23.

Embodiment 25 is a method of treating or preventing a metabolicdisorder, comprising administering to a subject in need thereof aneffective amount of the pharmaceutical composition according to any ofEmbodiments 22 and 23.

Embodiment 26 is a method according to Embodiment 25, wherein themetabolic disorder is selected from the group consisting of type 2diabetes, elevated glucose levels, elevated insulin levels, obesity,dyslipidemia, diabetic nephropathy, myocardial ischemic injury,congestive heart failure, or rheumatoid arthritis.

Embodiment 27 is a method according to Embodiment 25 or 26, wherein thepharmaceutical composition is administered to the subject subcutaneouslyor intravenously.

Embodiment 28 is a method of treating a metabolic disorder selected fromthe group consisting of type 2 diabetes, elevated glucose levels,elevated insulin levels, obesity, dyslipidemia, diabetic nephropathy,myocardial ischemic injury, congestive heart failure, or rheumatoidarthritis, in a subject in need thereof, the method comprisingsubcutaneously or intravenously administering to the subject atherapeutically effective amount of a pharmaceutical compositioncomprising a fusion protein comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 5, 25-30, 40, 55-60, and 70 anda pharmaceutically acceptable carrier.

Embodiment 29 is a method of treating a metabolic disorder selected fromthe group consisting of type 2 diabetes, elevated glucose levels,elevated insulin levels, obesity, dyslipidemia, diabetic nephropathy,myocardial ischemic injury, congestive heart failure, or rheumatoidarthritis, in a subject in need thereof, the method comprisingadministering to the subject a therapeutically effective amount of apharmaceutical composition comprising a fusion protein comprising theamino acid sequence of SEQ ID NO: 60 and a pharmaceutically acceptablecarrier.

Embodiment 30 is a method of any one of Embodiments 25 to 29, whereinthe pharmaceutical composition is administered to the subjectintravenously or subcutaneously.

Embodiment 31 is a fusion protein of any one of Embodiments 1 to 15 foruse in treating or preventing a metabolic disorder selected from thegroup consisting of type 2 diabetes, elevated glucose levels, elevatedinsulin levels, obesity, dyslipidemia, diabetic nephropathy, myocardialischemic injury, congestive heart failure, or rheumatoid arthritis.

EXAMPLES

The following examples of the invention are to further illustrate thenature of the invention. It should be understood that the followingexamples do not limit the invention and that the scope of the inventionis to be determined by the appended claims.

Example 1: Design of Fusion Molecules Comprising GDF15-Effect of GDF15Truncations

Like other TGFβ family members, GDF15 is synthesized as apre-pro-protein that forms a dimer in the endoplasmic reticulum andundergoes furin cleavage to produce secreted mature GDF15 (amino acids197-308). The secreted mature GDF15 homodimer is about 25 k Daltons, andeach monomer has the potential to form up to 4 intramolecular disulfidebonds with a single intermolecular disulfide linking the homodimercomponents.

The crystal structure of GDF15 was determined in the invention and isdepicted in FIGS. 1A and 1B. The crystal structure shows that theC-terminus of the mature GDF15 is buried in the dimer interface, whilethe N-terminus is exposed. This exposed terminus allows for the linkageof fusion proteins, such as half life extension proteins, to theN-terminus of GDF15.

The crystal structure also depicts the novel disulfide paring pattern ofGDF15 cysteine residues. While TGFβ1 has C1-C3 and C2-C7 pairing (i.e.,pairing between its first and third cysteine residues as well as betweenits second and seventh cysteine residues), GDF15 has C1-C2 and C3-C7pairing (see FIGS. 1A and 1B). This unique disulfide pairing results ina loop formed by the C1-C2 pairing that is located at the N-terminus ofthe protein and away from the cysteine knot that contains otherdisulfide bonds. The structure predicts that the N-terminus of GDF15 maynot be critical for dimer formation or overall protein folding, and thatGDF15 and N-terminal fusion molecules thereof may be tolerable toN-terminal deletions that delete C1 and C2, residues within the C1-C2loop, or even residues C-terminal to C2.

Example 2: Design of Fusion Molecules Comprising GDF15 —Effect of theLinker

Different linkers between the HSA molecule and the GDF15 molecule wereevaluated. Both flexible linkers, containing the sequence (GGGGS)n (SEQID NO: 129), and structured linkers, containing the sequence (AP)n (SEQID NO: 144) or (EAAAK)n (SEQ ID NO: 130), wherein n is 2 to 20, wereevaluated.

Fusion proteins comprising the different linkers were compared for theirbiophysical properties, their effect on the efficacy of food intake inlean mice, their mouse pharmacokinetic (PK) values, and their ex vivostability in human blood. The results of tested linker variants areshown in Table 1. The molecule comprising SEQ ID NO: 31, which containedthe (EAAAK)8 (SEQ ID NO: 138) linker, showed aggregation by HPLC. Theremaining seven linker variants in Table 1 demonstrated no aggregation.

TABLE 1 Summary of linker variant analysis SEQ Good Ex vivo ID Aggre-Mouse stability in NO* Linker of gation PK (WT) human blood 25AS(GGGGS)₂GT No Yes Yes 5 GS(GGGGS)₄ No Yes Yes 26 AS(GGGGS)₈GT No YesYes 27 AS(AP)₅GT No Yes Yes 28 AS(AP)₁₀GT No Yes Yes 29 AS(AP)₂₀GT NoYes Yes 30 AS(EAAAK)₄GT No Yes Yes 31 AS(EAAAK)₈GT YES Not Not testedtested *-6xHis tag was attached at the N-terminus for purificationpurpose

Linker stability was also evaluated for these variants by in vivostudies in mice and by ex vivo stability studies in human whole bloodand plasma samples. Two forms of detection were used to analyze theresults from these studies. An immunoassay with anti-GDF15 capture andanti-HSA detection antibody pairs was used to evaluate how intact thelinker was by measuring the presence of both molecules on either side ofthe linker. A broader picture of the whole-molecule integrity wasanalyzed by liquid chromatography-mass spectrometry (LC-MS) analysisusing different surrogate peptide sequences from both HSA and GDF15 .The immunoassay demonstrated a stable PK profile for all of the linkervariants and no loss of spiked plasma sample concentration for any ofthe linker variants observed over 48 hours. The LC-MS results wereconsistent with the immunoassay showing that the surrogate peptides fromdifferent parts of the HSA and GDF15 molecules were intact. The PKprofile of the linker variants analyzed by LC-MS using surrogatepeptides showed a similar trend for different linker variants, wherethey all had detectable levels at day 7. All the variants in Table 1except for SEQ ID 31 had desirable biophysical properties and PK values.

The linker variants were evaluated for their in vivo activity bycarrying out food intake studies in lean mice. Table 2 shows theinfluence of the linker variants on the efficacy of the fusion proteinin decreasing food intake. There was a clear influence of the linker onthe efficacy. With regard to the flexible (GGGGS)n (SEQ ID NO: 129)linkers, an increase in the linker length from 2 to 4 to 8 dramaticallyincreased the fusion protein efficacy. For the more rigid (AP)n (SEQ IDNO: 144) linkers, the trend was less obvious, suggesting that the degreeof freedom of the GDF15 molecule within the fusion protein plays acritical role in its efficacy.

TABLE 2 Effect of the linker on the in vivo efficacy of HSA-GDF15 fusion proteins in lean mice SEQ ID % Decrease in NO* Linkerfood intake (mean) 25 AS(GGGGS)₂GT 28.8 5 GS(GGGGS)₄ 40.5 26AS(GGGGS)₈GT 60.7 27 AS(AP)₅GT 48.2 28 AS(AP)₁₀GT 66.2 29 AS(AP)₂₀GT55.1 30 AS(EAAAK)₄GT 51.9 *-6xHis tag was attached at the N-terminus forpurification purpose

Example 3: Design of Fusion Molecules Comprising GDF15 —Effect of HSAMutations

Recombinant proteins with the half life extension protein human serumalbumin fused to the N-terminus of GDF15 through a linker were designed.This design should allow for the GDF15 dimerization interface to remainunperturbed and allow for the formation of the native inter-chaindisulfide linkages, resulting in a GDF15 homodimer with HSA fusionextended from each GDF15 arm. With this approach, only a single gene isrequired to generate the HSA-GDF15 homodimer.

Native human serum albumin protein contains 35 cysteine (Cys, C)residues that form 17 disulfide bonds, with the Cys-34 residue being theonly free cysteine in the molecule. This free Cys-34 has been shown tofunction as a free radical scavenger, by trapping multiple reactiveoxygen species (ROS) and reactive nitrogen species (RNS). This free Cyswas thus mutated to minimize the risk of heterogeneity due to oxidation.

The free cysteine at position 34 of HSA was mutated to either serine oralanine, and the GDF15 fusion molecules with either a HSA(C34S) or aHSA(C34A) mutation were analyzed. Both of the molecules were purifiedusing a three-step purification method: (i) ion-exchange chromatography,(ii) hydrophobic interaction chromatography, and (iii) size-exclusionchromatography. When they were first generated, HPLC analysis showedthat both molecules were pure and aggregation-free (Table 3).

However, two weeks after its generation, the fusion protein containingthe HSA(C34A) mutation (comprising SEQ ID NO: 48) showed aggregation byHPLC, while the fusion protein containing the HSA(C34S) mutation (SEQ IDNO: 40) remained aggregation-free after four weeks.

TABLE 3 The influence of mutating HSA C34 on fusion protein aggregation% aggregation % aggregation HSA when 2 weeks post SEQ ID NO mutationpurified purification 40 C34S 0 0 48 C34A 0 33.29

Example 4: Protease Cleavage Propensity on GDF15

It was observed by the inventors that the arginine residue at amino acidposition 198 of GDF15 (R198) is susceptible to protease degradationwithin the HSA-GDF15 fusion molecules. Such degradation results in aheterogeneous population and is undesirable for therapeuticcompositions. The cleavage can be prevented by a protease inhibitorcocktail. Purification methods were investigated for the removal of theprotease. Table 4 lists the two types of HSA affinity columns that weretested for purification of HSA-GDF15 fusion proteins, as measured byHPLC. At the time of purification, the HSA-GDF15 fusion proteinspurified by both methods were 100% pure and intact. At lowconcentrations (2-5 mg/ml), proteins purified by both methods remainedintact for the entire test period of 4 weeks. However, at highconcentrations (40-50 mg/ml), only the antibody-based HSA resin(CaptureSelect) produced protease-free proteins that remain intact forthe entire 4 week test period. The HSA-ligand-based resin (Albupure)generated proteins that were intact initially but demonstrateddegradation over time when stored at high concentrations. Adding aprotease inhibitor cocktail (PI) and EDTA completely arrested thedegradation of the high concentration HSA-GDF15 fusion protein batchpurified using the Albupure resin. Thus, the purification method plays acritical role in generating a stable therapeutic composition.Corresponding degradation was not observed in vivo or ex vivo,suggesting that once the therapeutic composition has been madeprotease-free, degradation of the fusion proteins is not an issue invivo. Therefore, purification methods that can effectively removepotential proteases during production, such as those using theCaptureSelect resin, are key to successfully manufacturing GDF15therapeutics that are homogenous, intact and stable.

TABLE 4 Protease cleavage of the HSA-GDF15 fusion proteins can beeliminated by sample purification methods % degraded population of HSA-Condition GDF15 (SEQ ID 60) +PI/ WEEK WEEK WEEK WEEK Concentration ResinEDTA 0 1 2 3 WEEK 4 Low Capture No 0 0 0 0 0 Select Low Albupure No 0 00 0 0 High Capture No 0 0 0 0 0 Select High Albupure No 0 3.04 9.4711.57 13.89 High Capture Yes 0 0 0 0 0 Select High Albupure Yes 0 0 0 00

Example 5: N-Terminal Deletion Variants of GDF15

The GDF15 crystal structure depicted in FIGS. 1A and 1B predicts thatthe N- terminus of GDF15 involved in the deletion variants is notcritical for dimer formation and overall protein folding. It alsopredicts that such N-terminal deletions should not affect any potentialreceptor interaction. HSA-GDF15 fusion proteins comprising variousdeletions of the N terminal of GDF15 were tested for in vivo activity.

GDF15 N-terminal deletion variants were designed that removed theprotease cleavage site at GDF15 (R198). Immediately following the R198residue, there is a potential deamidation site at residues N199-G200,and substrate deamidation is also not favored in therapeuticcompositions. GDF15 N-terminal deletions can remove both the proteolyticcleavage site and the deamidation sites simultaneously. The resultingGDF15 deletion variants that were incorporated into fusion proteins withHSA included GDF15 (201-308; SEQ ID NO: 8), GDF15 (202-308; SEQ ID NO:9), and GDF15 (211-308; SEQ ID NO: 11). In vivo studies in mice showedthat the N-terminal deletion variants of GDF15 are still active inreducing food intake (FIG. 17 ). The experimental results confirmed thatsuch GDF15 N-terminal deletion variants express properly, formappropriate dimers, and are active in vivo.

Example 6: Inactive Mutants of GDF15

Table 5 lists twelve mutants of GDF15 that were made to eliminate GDF15in vivo activity and identify the functional epitope of GDF15. Themutants include five single mutants, two double mutants, and five triplemutants. HSA-GDF15 fusion proteins comprising these mutations werecharacterized for their biophysical properties and activities (Table 5).Out of the 12 mutants, one did not express and four formed aggregatesover time, indicating that the mutations interrupt protein folding andbiophysical properties. Of the remaining seven mutants, four of themcontained a single mutation of GDF15, and these mutants were tested inmice for food intake reduction compared to wild type. Three of thesingle mutants (I89R, I89W and W32A) lost in vivo activity, while theremaining mutant (Q60W) is as active as the wild type. These resultsindicated that the I89R, I89W or W32A mutation interrupts theinteraction of the receptor/co-receptor with GDF15, suggesting that thefunctional epitopes of GDF15 are around residues I89 and W32. Thenumbering of the mutation is based on the mature GDF15 present in fusionprotein, e.g., “1” refers to the 1^(st) amino acid of the mature GDF15(SEQ ID NO: 6) and “89” refers to the 89^(th) amino acid of the matureGDF15 protein.

TABLE 5 Summary of the biophysical properties and activities of fusionproteins comprising GDF15 mutants SEQ ID Mutations NO in GDF15Biophysical properties Activities 5 Wild type Expresses well, stableWild type activity 64 I89R Expresses well, stable Complete loss ofactivity 65 I89W Expresses well, stable Complete loss of activity 66L34A, S35A, R37A Expresses well, stable 67 V87A, I89A, L98A Expresseswell, unstable 68 L34A, S35A, I89A Expresses well, stable 69 V87A, I89AExpresses well, stable 70 Q60W Expresses well, stable As active as wildtype 71 W32A Expresses well, stable Complete loss of activity 72 W29AExpresses well, unstable 73 Q60A, S64A, R67A Expresses well, unstable 74W29A, Q60A, I61A Does not express 75 W29A, W32A Expresses well, unstable*6xHis tag was attached at the N-terminus for purification purpose

Example 7: Expression and Purification Methods

Expression

For expression of 20 ml and greater, the expression was done using HEKExpi293™ cells grown in Expi293™ Expression media. The cells were grownat 37° C. while shaking at 125 RPM with 8% CO₂. The cells weretransfected at 2.5×10⁶ cells per ml using the Expi293™ Expression Kit.For each liter of cells transfected, 1 mg of total DNA was diluted in 25ml of Opti-MEM, and 2.6 ml of Expi293™ reagent was diluted in 25 ml ofOpti-MEM and incubated for 5 minutes at room temperature. The dilutedDNA and diluted Expi293 reagent were combined and incubated for 20minutes at room temperature. The DNA complex was then added to thecells. The cells were placed in the shaking incubator overnight. The dayafter transfection, 5 ml of Enhancer 1 from the kit was diluted into 50ml of Enhancer 2 from the kit, and the total volume of the two Enhancerswas added to the cells. The transfected cells were placed back into theincubator for 4 days until they were harvested. The cells wereconcentrated by centrifugation at 6,000 g for 30 minutes and thenfiltered with a 0.2 μm filter before the purification step.

The expression was also done in CHO cells. The plasmid was purified andcharacterized. Prior to transfection, 1 aliquot of 200 μg of plasmid DNAcontaining the coding region of HSA-GDF15 was linearized by restrictionenzyme digestion with Acl I. The digestion with this restrictionendonuclease ensures the removal of the ampicillin resistance gene. Twolinearized 15 μg DNA aliquots were transfected into two 1×10⁷ CHO cells(designated transfection pool A and B) using the BTX ECM 830 ElectroCell Manipulator (Harvard Apparatus, Holliston, MA). Cells wereelectroporated 3 times at 250 volts with 15 millisecond pulse lengthsand 5 second pulse intervals in a 4 mm gap cuvette. Transfected cellswere transferred to MACH-1+L-glutamine in a shake flask and incubatedfor 1 day. Transfection pool A and transfection pool B were centrifuged,resuspended in MACH-1+MSX, and transferred to shake flasks to incubatefor 6 days. Transfected HSA-protein fusion-producing cells fromtransfection pool A and transfection pool B were pooled and plated inmethylcellulose on day 8 post-electroporation.

Purification

Two-step purification using CaptureSelect resin and size exclusionchromatography was used. Cell supernatants from transiently transfectedExpi293™ cells were loaded onto a pre-equilibrated (PBS, pH 7.2) HSACaptureSelect column (CaptureSelect Human Albumin Affinity Matrix fromThermoFisher Scientific) at an approximate capacity of 10 mg protein perml of resin. After loading, unbound proteins were removed by washing thecolumn with 10 column volumes (CV) of PBS pH7.2. The HSA-GDF15 that wasbound to the column was eluted with 10 CV of 2 M MgCl₂ in 20 mM Tris, pH7.0. Peak fractions were pooled, filtered (0.2 μ), and dialyzed againstPBS pH 7.2 at 4° C. After dialysis, the protein was filtered (0.2 μagainand concentrated to an appropriate volume before loading onto a 26/60superdex 200 column (GE Healthcare). Protein fractions that eluted fromthe size exclusion chromatography (SEC) column with high purity(determined by SDS-PAGE) were pooled. The concentration of protein wasdetermined by the absorbance at 280 nm on a BioTek Synergy HTTMspectrophotometer. The quality of the purified proteins was assessed bySDS-PAGE and analytical size exclusion HPLC (SE-HPLC, Dionex HPLCsystem). Endotoxin levels were measured using a LAL assay (Pyrotell®-T,Associates of Cape Cod).

Two-step purification using Albupure resin and SEC was also used.HSA-GDF15 fusion proteins were purified at room temperature usingAlbuPure resin (ProMetic BioSciences Ltd) which utilizes an immobilizedsynthetic triazine ligand to selectively bind HSA. The expressionsupernatants were applied to the AlbuPure resin. The resin was thenwashed, first with 4 CV PBS pH 7.2 followed by 4 CV of 50 mM Tris pH8.0, 150 mM NaCl buffer. The HSA-GDF15 that was bound to the column waseluted with 4 CV of PBS pH 7.2 buffer containing 100 mM Na Octanoate.The protein-containing fractions were concentrated to a 10 mL volumeusing a 30,000 kDa molecular weight cutoff spin concentrator (Amicon)and then applied to a 26/60 Superdex S200 pg column (GE) that wasequilibrated in PBS pH 7.2 buffer. SEC fractions containing HSA-GDF15homodimer were identified via SDS-PAGE and pooled for analysis. Theprotein purities were assessed by SDS-PAGE and SE-HPLC.

The Examples 8-14, and 19 involve characterization of an exemplaryfusion protein of the invention, which has the amino acid sequence ofSEQ ID NO: 60. This fusion protein is a fully recombinant protein thatexists as a homodimer of a fusion of HSA with the mature human GDF15through a 42-amino acid linker consisting of glycine and serineresidues, GS-(GGGGS)8 (SEQ ID NO: 12). The predicted molecular weight ofthis fusion protein is 162,696 Daltons, and the single native freecysteine at position 34 of HSA has been mutated to serine. Thisparticular HSA-GDF15 fusion protein will be referred to simply as “FP1”in the following examples, for simplicity. A 6xHis-tagged variant of FP1(6xHis-FP1, SEQ ID NO: 26), containing an AS-(GGGGS)x 8-GT (SEQ ID NO:141) linker, was used for comparison in some of the following examples.

Example 8: Effects of FP1 on the Food Intake of C57B1/6 Mice

The purpose of this experiment was to demonstrate the dose-responsiveeffect of FP1 on the inhibition of food intake in C57B1/6 mice.

Male C57B1/6 mice were acclimated for a minimum of 72 hours in BioDAQcages. Mice were then grouped based on food intake in the previous 24hours into six groups of eight. Between 4:00 and 5:00 pm, animals wereweighed and dosed with vehicle or a composition comprising FP1 viasubcutaneous injection. The change in food weight for each cage wasrecorded continuously by the BioDAQ system for a period of 48 hoursafter the injections. 6xHis-FP1 was used for comparison in this study.

The results (FIG. 2 and Table 6) were expressed as an average ofcumulative food intake for a given time interval. The results indicatedthat subcutaneous administration of FP1 to C57BL/6 mice significantlyinhibited food intake relative to vehicle-treated animals at all dosesand time points tested. 6xHis-FP1 reduced food intake at the 8 nmol/kgdose.

TABLE 6 Effects of subcutaneous administration of FP1 on food intake inC57BL/6 mice; Cumulative food intake at 12, 24 and 48 hours postadministration is shown Cumulative Food Intake (g) Treatment 12 hours 24hours 48 hours PBS 3.7 ± 0.2   4.3 ± 0.1   8.4 ± 0.3   FP1, 1 nmol/kg2.9 ± 0.1*   3.4 ± 0.2**  7.0 ± 0.3**  FP1, 4 nmol/kg 2.3 ± 0.2**** 3.1± 0.1**** 6.6 ± 0.3***  FP1, 8 nmol/kg 2.1 ± 0.2**** 3.2 ± 0.1***  6.4 ±0.2**** FP1, 16 nmol/kg 1.7 ± 0.2**** 2.7 ± 0.2**** 6.2 ± 0.3****6xHis-FP1, 8 nmol/kg 1.8 ± 0.2**** 2.6 ± 0.2**** 6.0 ± 0.2**** Data areexpressed as Mean ± SEM. *p ≤ 0.05, versus PBS; **p ≤ 0.01, versus PBS;***p ≤ 0.001, versus PBS; ****p ≤ 0.0001, versus PBS One-WayANOVA-Tukey's multiple comparisons test; n = 8/group

Example 9: Effects of FP1 on Food Intake in Sprague Dawley Rats

The purpose of this experiment was to demonstrate the dose-responsiveeffect of FP1 on the inhibition of food intake in Sprague Dawley rats.

Male Sprague-Dawley rats were acclimated for a minimum of 72 hours inthe BioDAQ cages. Rats were then grouped based on food intake in theprevious 24 hours into six groups of eight. Between 4:00 and 5:00 pm,animals were weighed and dosed with vehicle or a composition comprisingthe fusion protein via subcutaneous injection. The change in food weightfor each cage was recorded continuously by the BioDAQ system, for aperiod of 48 hours after the injections. 6xHis-FP1 was used forcomparison in this study.

The results are shown in FIG. 3 and Table 7. Subcutaneous administrationof FP1 inhibited food intake at doses of 2.5 nmol/kg and 10 nmol/kgcompared to vehicle-treated animals. The inhibition reached statisticalsignificance only with the highest dose tested (10 nmol/kg) at 24 and 48hours post-administration. 6xHis-FP1 reduced food intake at the 8nmol/kg dose, and the effect was significant at 24 and 48 hours.

TABLE 7 Effects of subcutaneous administration of FP1 on food intake inSprague-Dawley rats; cumulative food intake at 12, 24 and 48 hours postadministration is shown Cumulative Food Intake (g) Treatment 12 hours 24hours 48 hours PBS 20.6 ± 1.3 25.3 ± 1.3  49.1 ± 2.1  FP1, 0.1 nmol/kg23.3 ± 1.4 26.9 ± 0.8  52.8 ± 1.3  FP1, 0.5 nmol/kg 22.6 ± 1.7 25.1 ±1.0  48.3 ± 1.8  FP1, 2.5 nmol/kg 20.0 ± 1.4 22.0 ± 1.0  44.6 ± 1.4 FP1, 10 nmol/kg 18.7 ± 0.9 19.9 ± 1.0*  39.9 ± 2.3*  6xHis-FP1, 8nmol/kg 17.0 ± 1.5 18.8 ± 1.4** 38.4 ± 2.5** Data are expressed as Mean± SEM. *p ≤ 0.05, versus PBS; **p ≤ 0.01, versus PBS One-WayANOVA-Tukey's multiple comparisons test; n = 8/group

Example 10: Effects of FP1 on Glucose Homeostasis and Body Weight inDiet-Induced Obese (DIO) Mice

The purpose of this experiment was to evaluate the effects of FP1 onfood intake, body weight, and glucose homeostasis throughout two weeksof treatment in DIO C57B1/6 mice.

Male DIO mice were weighed, and FP1 was dosed subcutaneously at 2 mL/kgevery three days (q3d) at Day 0, 3, 6, 9, and 12. The vehicle androsiglitazone treatment groups were dosed with PBS on a similar regimen.The control rosiglitazone was provided in the diet at 0.015% ad libitum.Mouse and food weights were recorded daily. Glucose was measured using aglucometer (One Touch®Ultra®, Lifescan, Milpitas, Calif.). Fat and leanmass was quantitated in conscious mice by time-domain NMR (TD-NMR) usingthe Bruker Mini-Spec LF110. For an oral glucose tolerance test (OGTT),mice were fasted for 4 hours. Blood glucose was measured via tail snipat 0, 30, 60, 90, and 120 minutes post oral gavage administration of 2g/kg glucose at 10 mL/kg. Insulin was measured at 0, 30, and 90 minutespost glucose administration.

At the end of the study, the mice were euthanized via CO₂ inhalation,and a terminal blood sample was collected. Serum was placed into a 96well plate on wet ice and then stored at −80° C. The liver was removed,and the fat content relative to the total mass of liver sections wasassessed using TD-NMR with the Bruker Mini Spec mq60 according to themanufacturer's instructions.

The fasted homeostatic model assessment of insulin resistance (HOMA-IR)was calculated based on the product of fasted glucose (in mg/dL) andinsulin (in mU/L) divided by a factor of 405.

Treatment of DIO mice with FP1 q3d at 1 nmol/kg and 10 nmol/kg reducedbody weight (Table 8) and food intake (Table 9). The reductions reachedstatistical significance only at certain time points, as describedbelow.

FP1 decreased body weight at doses of 1 (from day 2 to 14) and 10nmol/kg (from day 1 to 14) in DIO mice (Table 8 and FIG. 4 ). Asignificant reduction in food intake was seen at days 1 and 2 of thestudy at the dose of 1 nmol/kg and at days 1, 8 and 9 at the 10 nmol/kgdose (Table 9).

TABLE 8 Body weight change (% of starting) during treatment with FP1 inDIO mice Treat- ment Vehicle FP1 (nmol/kg) Rosiglitazone Day n/a 0.1 110 10 mpk/day −2   0.1 ± 0.2 −0.1 ± 0.4   0.6 ± 0.4    0.2 ± 0.3  0.1 ±0.2  −1 −0.8 ± 0.3 −0.2 ± 0.3   0.4 ± 0.3  −0.3 ± 0.4  −0.9 ± 0.2    0  0.0 ± 0.0   0.0 ± 0.0   0.0 ± 0.0    0.0 ± 0.0  0.0 ± 0.0  1   0.1 ±0.2 −0.1 ± 0.4 −1.5 ± 0.5  −2.8 ± 0.4* 1.9 ± 0.2  2   0.0 ± 0.3 −0.8 ±0.5 −3.2 ± 0.4* −3.1 ± 0.5* 2.2 ± 0.5  3 −0.2 ± 0.4 −0.4 ± 0.4 −3.2 ±0.6* −4.0 ± 0.7* 2.6 ± 0.5  4 −0.4 ± 0.5 −0.7 ± 0.5 −3.8 ± 0.6* −4.4 ±0.8* 3.0 ± 0.5* 5 −0.5 ± 0.4 −1.0 ± 0.3 −4.0 ± 0.5* −4.6 ± 0.9* 3.8 ±0.6* 6 −0.4 ± 0.6 −0.5 ± 0.5 −3.8 ± 0.4* −5.6 ± 0.9* 4.0 ± 0.7* 7 −0.3 ±0.5 −0.5 ± 0.5 −4.3 ± 0.6* −6.1 ± 0.9* 5.5 ± 0.8* 8 −0.6 ± 0.5 −0.5 ±0.5 −4.2 ± 0.6* −6.7 ± 1.1* 6.1 ± 0.9* 9 −0.4 ± 0.5 −0.2 ± 0.5 −4.5 ±0.6* −7.2 ± 1.2* 7.0 ± 1.0* 10 −0.3 ± 0.5   0.3 ± 0.5 −4.4 ± 0.8* −7.8 ±1.2* 7.8 ± 1.1* 11 −0.5 ± 0.5   0.5 ± 0.4 −4.4 ± 0.8* −7.9 ± 1.4* 8.2 ±1.2* 12 −0.8 ± 0.6   0.8 ± 0.5 −4.3 ± 1.0* −7.9 ± 1.5* 8.5 ± 1.2* 13−0.8 ± 0.6   0.5 ± 0.5 −3.9 ± 0.9* −8.7 ± 1.6* 8.6 ± 1.3* 14 −1.7 ± 0.6−0.4 ± 0.5 −4.6 ± 1.0* −9.0 ± 1.7* 7.6 ± 1.3* Data are expressed as Mean± SEM. n = 8 per group. *= p < 0.05, compared to that of the vehicletreated group.

TABLE 9 Daily food intake (gm) during treatment with FP1 in DIO miceTreatment Vehicle FP1 (nmol/kg) Rosiglitazone Day n/a 0.1 1 10 10mpk/day −2 2.9 ± 0.1 3.0 ± 0.2 2.9 ± 0.1 2.8 ± 0.1 2.8 ± 0.1 0 3.0 ± 0.13.0 ± 0.1 2.8 ± 0.1 2.8 ± 0.1 3.0 ± 0.1 1 2.9 ± 0.1 3.1 ± 0.1 2.1 ± 0.2*1.5 ± 0.1* 3.6 ± 0.1* 2 3.0 ± 0.1 3.0 ± 0.1 2.3 ± 0.1* 2.6 ± 0.2 3.9 ±0.2* 3 2.8 ± 0.1 3.0 ± 0.1 2.6 ± 0.1 2.4 ± 0.2 3.5 ± 0.1* 4 2.3 ± 0.12.6 ± 0.1 2.2 ± 0.1 2.3 ± 0.1 3.3 ± 0.1* 5 2.8 ± 0.1 3.0 ± 0.1 2.8 ± 0.12.6 ± 0.1 3.7 ± 0.2* 6 2.8 ± 0.1 3.1 ± 0.1 2.9 ± 0.1 2.4 ± 0.2 3.7 ±0.2* 7 2.7 ± 0.1 2.9 ± 0.1 2.6 ± 0.1 2.4 ± 0.1 3.8 ± 0.2* 8 2.7 ± 0.12.9 ± 0.1 2.6 ± 0.1 2.1 ± 0.1* 3.5 ± 0.2* 9 3.0 ± 0.1 3.3 ± 0.1 2.8 ±0.1 2.5 ± 0.2* 4.3 ± 0.2* 10 2.6 ± 0.1 3.0 ± 0.1 2.6 ± 0.1 2.2 ± 0.1 3.6± 0.1* 11 2.9 ± 0.1 3.1 ± 0.1 2.7 ± 0.2 2.6 ± 0.2 3.4 ± 0.1* 12 2.7 ±0.1 3.1 ± 0.1 3.0 ± 0.1 3.0 ± 0.2 3.6 ± 0.2* 13 2.6 ± 0.1 2.8 ± 0.1 2.8± 0.1 2.2 ± 0.2 3.2 ± 0.1* Data are expressed as Mean ± SEM. n = 8 pergroup. *= p < 0.05, compared to that of the vehicle treated group.

In an OGTT performed on day 14 of the study, FP1 significantly loweredglucose levels compared to vehicle-treated animals at all time pointsafter time 0 at all three doses tested (Table 10). This was furtherquantitated as total area under the curve (AUC) and delta AUC, whichwere significantly lower compared to vehicle for all three doses tested(Table 10 and FIGS. 5A and 5B).

TABLE 10 Blood glucose (mg/dL) levels during an OGTT after fourteen daysof q3d dosing of FP1 in DIO mice Time after Glucose Challenge Total AUCDelta AUC Dose (min) (mg/dL/120 (mg/dL/120 Treatment (nmol/kg) 0 30 6090 120 min) min) Vehicle NA 209.6 ± 399.4 ± 338.5 ± 297.5 ± 269.3 ±38244.4 ± 13089.4 ±  16.5 46.4 46.3 38.0 36.6 4308.2 3573.0 FP1 0.1196.6 ± 319.0 ± 241.6 ± 196.0 ± 184.0 ± 28408.1 ± 4885.5 ± 7.6 21.5 14.412.8 6.3* 1236.1* 955.1* 1 164.0 ± 251.4 ± 227.6 ± 191.9 ± 180.6 ±25295.6 ± 5615.6 ± 7.2 13.0 12.6 9.4* 13.1 1026.7* 647.6* 10 146.6 ±195.6 ± 197.1 ± 172.1 ± 174.9 ± 21768.8 ± 4211.4 ± 5.9 14.5 6.9* 6.3*13.8 603.0* 425.8* Rosi- 10 130.8 ± 199.1 ± 204.4 ± 184.1 ± 169.1 ±22115.6 ± 6425.6 ± glitazone mpk/day 6.0 12.9 8.5* 10.9 5.7* 671.9*599.6 Data are expressed as Mean ± SEM. n = 8 per group. *= p < 0.05,compared to that of the vehicle treated group.

Fed blood glucose levels were measured at the start (day 0), at day 7and at day 13 of the study (Table 11 and FIG. 6 ). FP1 decreased bloodglucose in a statistically significant manner at doses of 1 nmol/kg and10 nmol/kg on day 13 of the study.

TABLE 11 Fed blood glucose during treatment of DIO mice with q3dtreatment of FP1 Dose Time after start of treatment (days) Treatment(nmol/kg) 0 7 13 Vehicle NA 177.6 ± 10.9 173.6 ± 10.6 225.3 ± 23.0 FP10.1 174.1 ± 8.5 171.8 ± 16.3 196.4 ± 8.1 1 167.5 ± 7.8 135.1 ± 11.2165.3 ± 10.3* 10 165.3 ± 13.7 145.3 ± 4.9 153.8 ± 7.5* Rosiglitazone 10mpk/day 189.6 ± 17.3 122.6 ± 8.1* 154.8 ± 6.5* Data are expressed asMean ± SEM. n = 8 per group. *= p < 0.05, compared to that of thevehicle treated group.

Plasma insulin levels during the OGTT were significantly higher for FP1than for the corresponding vehicle group for a 0.1 nmol/kg dose at 30minutes, and lower at the 1 and 10 nmol/kg doses at the same time point(Table 12). The insulin excursion during the OGTT, as measured by totalAUC, was higher than the vehicle group for the 0.1 nmol/kg dose of FP1(Table 12), and lower at the 1 and 10 nmol/kg dose. In both cases,statistical significance was reached only at the lowest dose. At the 90minute time point, mice treated with 1 and 10 nmol/kg of FP1 had lowerinsulin levels; however, this effect did not achieve statisticalsignificance. HOMA-IR, used as a measure of insulin sensitivity, wasmeasured on day 14 of the study. At this time point, FP1 decreasedHOMA-IR, or improved insulin sensitivity, at 10 nmol/kg (Table 13 andFIG. 7 ).

TABLE 12 Plasma insulin (pg/mL) levels during an OGTT after fourteendays of q3d dosing of FP1 in DIO mice Dose Time after Glucose Challenge(min) Total AUC Treatment (nmol/kg) 0 30 90 (pg/mL/90 min) Vehicle NA6096.0 ± 774.3 14660.0 ± 3031.2 5034.4 ± 405.1  902151.9 ± 143123.2 FP10.1 7861.0 ± 779.5 33825.8 ± 7902.0* 6494.4 ± 797.0 1834808.8 ±381276.1* 1 4808.1 ± 795.8 13061.8 ± 2226.3 3443.5 ± 342.5  763218.1 ±119766.8 10 3478.3 ± 634.6  7147.0 ± 823.8 2958.0 ± 414.0  462528.8 ±44653.1 Rosiglitazone 10 mpk/day 2965.6 ± 524.2  2203.1 ± 193.5* 1180.0± 57.1  179025.0 ± 9970.7 Time after Glucose Challenge (min) Data areexpressed as Mean ± SEM. n = 8 per group. *= p < 0.05, compared to thatof the vehicle treated group.

TABLE 13 Fasted HOMA-IR in DIO mice after fourteen days of q3d treatmentof FP1 Treatment Dose (nmol/kg) HOMA-IR Vehicle NA  93.5 ± 15.3 FP1 0.1112.4 ± 14.6 1  56.7 ± 9.7 10  37.7 ± 8.2* Rosiglitazone 10 mpk/day 27.3 ± 4.7* Data are expressed as Mean ± SEM. n = 8 per group. *= p <0.05, compared to that of the vehicle treated group.

The magnitude of weight loss achieved by day 13 did not result inmeasurable changes in absolute fat mass or percent fat mass at any dose(Table 14). At the 10 nmol/kg dose, there was a significant decrease inabsolute lean mass. This decrease was not observed when expressed aspercent lean mass. Liver weights were measured during terminal necropsyon day 15 of the study (Table 15). FP1 decreased absolute liver weightand liver weight as a percentage of body weight at the 10 nmol/kg dose.A decrease was observed at the 1 nmol/kg dose, but this did not reachstatistical significance for either parameter. Liver fat was measured ona biopsy by NMR (Table 16). FP1 fusion protein decreased hepatic fatcontent, expressed as a percentage of liver biopsy weight, at 1 and 10nmol/kg doses. The reduction was significant at the higher dose.

TABLE 14 Body composition after thirteen days of treatment with FP1 q3din DIO mice Fat Mass Lean Mass Dose Fat Mass Lean Mass (% of (% ofTreatment (nmol/kg) (g) (g) body) body) Vehicle NA 11.6 ± 0.5 29.6 ± 0.423.4 ± 0.8 59.6 ± 0.9 FP1 0.1 12.2 ± 0.4 29.7 ± 0.4 24.1 ± 0.6 58.5 ±0.6 1 12.1 ± 0.3 28.0 ± 0.4 25.1 ± 0.3 58.3 ± 0.6 10 10.6 ± 0.3 27.7 ±0.4* 23.1 ± 0.3 60.6 ± 0.6 Rosiglita- 10 mpk/day 14.9 ± 0.6* 30.0 ± 0.527.2 ± 0.4* 55.0 ± 0.6* zone Data are expressed as Mean ± SEM. n = 8 pergroup. *= p < 0.05, compared to that of the vehicle treated group.

TABLE 15 Liver weight after fifteen days of treatment with FP1 q3d inDIO mice Liver Weight Treatment Dose (nmol/kg) Liver weight (g) (% ofbody) Vehicle NA 2.6 ± 0.1 5.4 ± 0.2 FP1 0.1 2.9 ± 0.2 5.8 ± 0.2 1 2.2 ±0.1 4.6 ± 0.2 10 1.9 ± 0.1* 4.2 ± 0.1* Rosiglitazone 10 mpk/day 2.5 ±0.2 4.6 ± 0.2 Data are expressed as Mean ± SEM. n = 8 per group. *= p <0.05, compared to that of the vehicle treated group.

TABLE 16 Liver fat content measured after fifteen days of treatment withFP1 q3d in DIO mice Treatment Dose (nmol/kg) Fat (%) Vehicle NA 27.3 ±2.1 FP1 0.1 26.0 ± 1.4 1 22.5 ± 1.4 10 17.8 ± 1.6* Rosiglitazone 10mpk/day 25.9 ± 0.8 Data are expressed as Mean ± SEM. n = 8 per group. *=p < 0.05, compared to that of the vehicle treated group.

Example 11: Effects of FP1 on Blood Glucose Levels and Body Weight inob/ob Mice

The purpose of this experiment was to evaluate the effects of FP1 onbody weight and blood glucose levels over eight days of treatment inobese, hyperglycemic, leptin-deficient ob/ob mice.

Male ob/ob mice were weighed and FP1 was administered subcutaneously at2mL/kg every three days (q3d) at Day 0, 3 and 6. Mouse and food weightswere recorded daily. Glucose was measured daily using a glucometer. Atthe end of the study, mice were euthanized, and a terminal blood samplewas collected.

FP1, at the 1 nmol/kg dose, significantly decreased body weight(expressed as a percentage of starting body weight) in ob/ob micestarting at day 2 until day 8, relative to vehicle-treated mice. FP1, atthe 10 nmol/kg dose, decreased body weight (expressed as a percentage ofstarting body weight) in ob/ob mice starting at day 1 until day 8relative to vehicle-treated mice (Table 17 and FIG. 8 ).

TABLE 17 Body weight change (% of starting) during treatment with FP1q3d in ob/ob mice Treatment Vehicle FP1 Day n/a 1 nmol/kg 10 nmol/kg −6−2.5 ± 0.3 −2.8 ± 0.4 −3.6 ± 0.5 −5 −2.4 ± 0.3 −3.0 ± 0.5 −3.5 ± 0.5 −4−1.9 ± 0.2 −2.5 ± 0.4 −2.8 ± 0.3 −1 −0.3 ± 0.3   0.1 ± 0.2 −0.3 ± 0.2 0  0.0 ± 0.0   0.0 ± 0.0   0.0 ± 0.0 1   0.5 ± 0.2 −1.6 ± 0.2 −2.3 ± 0.5*2   1.1 ± 0.2 −1.8 ± 0.4* −2.0 ± 0.9* 3   1.3 ± 0.3 −2.5 ± 0.4* −3.1 ±1.1* 4   1.8 ± 0.3 −3.3 ± 0.5* −4.2 ± 1.3* 5   1.8 ± 0.4 −3.5 ± 0.5*−4.7 ± 1.5* 6   2.0 ± 0.5 −4.0 ± 0.7* −5.7 ± 1.6* 7   2.8 ± 0.6 −4.8 ±0.8* −6.3 ± 1.7* 8   3.5 ± 0.8 −4.5 ± 1.0* −6.1 ± 1.9* Data areexpressed as Mean ± SEM. n = 8 per group. *= p < 0.05, compared to thatof the vehicle treated group.

FP1, at the 10 nmol/kg dose, decreased fed blood glucose values in ob/obmice on study day 1 and 2 and from day 4 until day 8 relative tovehicle-treated mice. A reduction in in blood glucose was observed at 1nmol/kg; however, this effect did not reach statistical significance(Table 18 and FIG. 9 ).

TABLE 18 Fed blood glucose during treatment of ob/ob mice with FP1 q3dTreatment Vehicle FP1 Day n/a 1 nmol/kg 10 nmol/kg −6 463.6 ± 31.2 395.7± 52.0 463.6 ± 41.0 −5 554.9 ± 57.5 609.6 ± 53.6 552.9 ± 53.0 −4 502.9 ±41.6 517.9 ± 71.6 490.3 ± 54.4 −1 552.1 ± 45.1 567.2 ± 51.2 586.0 ± 42.60 468.8 ± 57.3 479.7 ± 61.8 437.0 ± 42.7 1 537.0 ± 42.8 439.0 ± 80.4336.2 ± 51.0* 2 511.7 ± 43.5 440.0 ± 74.3 293.7 ± 37.6* 3 447.8 ± 54.2369.6 ± 75.9 279.4 ± 38.8 4 516.3 ± 47.0 384.6 ± 84.0 261.2 ± 43.7* 5531.0 ± 47.1 410.9 ± 92.2 286.9 ± 55.8* 6 596.1 ± 45.1 451.1 ± 82.6301.9 ± 49.3* 7 566.7 ± 44.3 425.3 ± 86.9 246.7 ± 43.5* 8 509.9 ± 37.6337.8 ± 75.1 223.4 ± 34.1* Data are expressed as Mean ± SEM. n = 8 pergroup.

Example 12: Multispecies Pharmacokinetics

Mouse Pharmacokinetics

FP1 was administered to female C57B1/6 mice at a dose of 2 mg/kg IV andSC in PBS, pH 7. Blood samples were collected, serum was processed anddrug concentrations were measured up to 7 days following both routes ofadministration. The concentration of FP1 was determined using animmunoassay method. The serum drug concentration-time profile issummarized in Tables 19 and 20 and illustrated in FIG. 10 .

TABLE 19 Serum concentration (nM) of FP1 over time following a single SCadministration in C57Bl/6 female mice FPl-SC Dose Animal Animal AnimalAnimal Animal Average 56 Result 57 Result 58 Result 60 Result 63 ResultResult Std Timepoint (nM) (nM) (nM) (nM) (nM) (nM) Dev  4 hr 32.29950.735 42.766 32.407 23.018 36.245 10.698 24 hr 88.822 106.418 88.648103.841 80.346 93.615 11.093 72 hr 33.563 38.473 32.473 33.625 32.76934.181 2.451 96 hr 20.639 24.988 21.247 19.356 20.771 21.400 2.124 Day 75.399 6.919 7.234 5.994 5.637 6.237 0.803

TABLE 20 Serum concentration (nM) of FP1 over time following a single IVadministration in C57Bl/6 female mice FP1-IV Dose Animal Animal AnimalAnimal Animal Average 52 Result 53 Result 65 Result 66 Result 70 ResultResult Std Timepoint (nM) (nM) (nM) (nM) (nM) (nM) Dev  1 hr 240.419233.318 232.484 276.913 272.727 251.172 21.857 24 hr 86.823 95.77480.201 93.153 88.853 88.961 6.027 72 hr 33.634 37.447 33.108 41.68034.034 35.981 3.612 96 hr 22.666 20.588 19.458 33.718 20.361 23.3585.909 Day 7 7.401 5.606 4.896 8.556 4.205 6.133 1.803

Pharmacokinetic analysis revealed a terminal half-life of 1.67 and 1.57days for FP1 in C57B1/6 mice following SC and IV administration,respectively (Table 21). FP1 demonstrated a mean bioavailability of ˜71%following SC administration.

TABLE 21 Mean (±SD) pharmacokinetic parameters of FP1 following 2 mg/kgIV and SC administration in female C57Bl/6 mice CL or t_(1/2) CL/F VssC_(max) T_(max)* AUC_(0-last) AUC_(0-inf) Route (day) (ml/day/kg)(ml/kg) (ng/ml) (day) (day*ng/ml) (day*ng/ml) SC Mean 1.67 49.48 14994 138315 40734 (SD) (0.14) (4.791)  (1776)  (3851)  (4072) IV Mean 1.5735.00 69.03 40231 0.04 55263 57531 (SD) (0.19) (3.13) (5.8)  (3500) (4853)  (5416) Note: *Tmax (median)

Rat Pharmacokinetics

FP1 was administered to female Sprague Dawley rats at a dose of 2 mg/kgIV and SC in PBS, pH 7. Blood samples were collected, serum wasprocessed and drug concentrations were measured up to 7 days followingboth routes of administration. The concentration of FP1 was determinedusing an immunoassay method. The serum drug concentration-time profileis summarized in Tables 22 and 23 and illustrated in FIG. 11 .

TABLE 22 Serum concentration (nM) of FP1 over time following a single SCadministration in female Sprague Dawley rats. FP1-Group 3 (SC Dose)Animal Animal Animal Animal Animal Average 53 Result 55 Result 67 Result68 Result 69 Result Result Timepoint (nM) (nM) (nM) (nM) (nM) (nM) StdDev  4 hr 4.766 3.500 3.932 3.546 3.250 3.799 0.593 24 hr 45.118 53.19239.196 39.823 40.804 43.627 5.826 72 hr 18.900 24.102 23.124 23.71818.933 21.755 2.615 96 hr 12.193 14.333 14.185 14.669 11.256 13.3271.511 Day 7 2.805 2.821 2.447 3.438 2.358 2.774 0.426

TABLE 23 Serum concentration (nM) of FP1 over time following a single IVadministration in female Sprague Dawley rats FP1-Group 4 (IV Dose)Animal Animal Animal Animal Animal 51 52 57 64 66 Average Result ResultResult Result Result Result Timepoint (nM) (nM) (nM) (nM) (nM) (nM) StdDev  1 hr 43.620* 382.676 403.255 443.080 510.105 356.547 181.560 24 hr102.665* 142.661 139.066 124.528 126.425 127.069 15.728 72 hr 46.72060.105 67.090 59.257 70.423 60.719 9.127 96 hr 29.409 39.897 41.22542.258 48.074 40.173 6.779 Day 7 6.976 11.251 8.913 9.540 13.006 9.9372.298 *Repeat analysis confirmed results

Pharmacokinetic analysis revealed a terminal half-life of 1.34 and 1.51days for FP1 in Sprague Dawley rats following SC and IV administration,respectively (Table 24). FP1 demonstrated a mean bioavailability of ˜23%following SC administration.

TABLE 24 Mean (±SD) pharmacokinetic parameters of FP1 following 2 mg/kgIV and SC administration in Sprague Dawley rats CL or t_(1/2) CL/F VssC_(max) T_(max)* AUC_(0-last) AUC_(0-inf) Route (day) (ml/day/kg)(ml/kg) (ng/ml) (day) (day*ng/ml) (day*ng/ml) SC Mean 1.34 100.09  69871 19250 20112 (SD) (0.04) (3.97)  (417)  (794)  (820) IV Mean 1.51 24.7553.41 59000 0.04 83028 86525 (SD) (0.12) (8.53) (17.15) (25031) (20126)(20881) Note: *Tmax (median)

Monkey Pharmacokinetics

FP1 was administered to naive male cynomolgus monkeys (Macacafascicularis) at a dose of 1 mg/kg IV and SC in PBS, pH 7. Blood sampleswere collected, serum was processed and drug concentrations weremeasured up to 21 days following both routes of administration, usingimmunoassay bioanalysis. The serum drug concentration-time profile issummarized in Tables 25 and 26 and illustrated in FIG. 12 .

TABLE 25 Serum concentration (nM) of FP1 over time following a single SCadministration in cynomolgus monkeys as determined by immunoassay FP1(SC Dose) Animal Animal Animal Average Std Timepoint 110 (nM) 111 (nM)112 (nM) Result (nM) Dev Predose <LLOQ <LLOQ <LLOQ <LLOQ N/A  6 hr49.304 43.784 72.110 55.066 15.017  24 hr 93.368 71.958 96.863 87.39613.483  48 hr 107.689 97.509 115.144 106.781 8.853  72 hr 113.601104.190 104.449 107.414 5.360 120 hr 101.490 95.049 91.717 96.085 4.968168 hr 82.167 75.435 81.569 79.724 3.726 240 hr 71.033 56.732 59.26662.344 7.631 336 hr 44.380 42.758 42.571 43.236 0.995 432 hr 30.91129.445 32.839 31.065 1.702 528 hr 21.277 20.404 26.427 22.703 3.255

TABLE 26 Serum concentration (nM) of FP1 over time following a single IVadministration in cynomolgus monkeys as determined by immunoassay FP1(IV Dose) Animal Animal Animal Average Std Timepoint 104 (nM) 105 (nM)106 (nM) Result (nM) Dev Predose <LLOQ <LLOQ <LLOQ <LLOQ N/A  1 hr212.661 235.168 189.000 212.276 23.087  6 hr 190.315 185.331 183.575186.407 3.497  24 hr 141.743 155.943 146.487 148.058 7.229  48 hr111.765 126.105 120.744 119.538 7.246  72 hr 105.076 106.955 106.441106.157 0.971 120 hr 92.591 103.882 103.757 100.077 6.483 168 hr 71.36893.055 87.706 84.043 11.298 240 hr 71.554 65.093 65.685 67.444 3.572 336hr 46.184 38.961 41.696 42.280 3.647 432 hr 34.589 31.266 19.492 28.4497.933 528 hr 26.885 24.154 22.422 24.487 2.250

Pharmacokinetic analysis revealed a terminal half-life between 8.5 and9.2 days for FP1 in cynomolgus monkeys following SC and IVadministration, respectively with a mean bioavailability of ˜88%following SC administration (Table 27).

TABLE 27 Mean (±SD) pharmacokinetic parameters of FP1 following 1 mg/kgIV and SC administration in cynomolgus monkeys. CL or t_(1/2) CL/F VssC_(max) T_(max)* AUC_(0-last) AUC_(0-inf) Route (day) (ml/day/kg)(ml/kg) (ng/ml) (day) (day*ng/ml) (day*ng/ml) SC Mean 8.5 3.9 17776 3211030 256202 (SD) (1.5) (0.3)  (950) (11332) (20192) IV Mean 9.2 3.445.6 34001 0.04 239758 292206 (SD) (0.5) (0.1) (1.6)  (3698)  (6095)(11516) Note: *Tmax (median)

Immuno-affinity capture-LCMS analysis was used to quantitate theconcentration of intact dimer present in the serum of cynomolgus monkeysafter IV and SC administration (Tables 28 and 29 and FIGS. 13 and 14 ).Concentrations determined by this method were similar to concentrationsdetermined by the immunoassay (IA), demonstrating that FP1 circulates asan intact dimer, with no detectable metabolic liability in cynomolgusmonkeys.

TABLE 28 Serum concentration (ng/mL) of FP1 as an intact dimer over timefollowing a single IV administration in cynomolgus monkeys as determinedby immuno-affinity capture-LCMS analysis. Dimer Intact MS Data Day(Pooled Samples) IA Data (Average) 0.00 0 0 0.04 33347 34537 0.25 2968630328 1.00 28787 24089 2.00 17249 19449 3.00 16827 17272 5.00 1615916282 7.00 11124 13674 10.00 8746 10973 14.00 5328 6879 18.00 3857 462922.00 2252 3984

TABLE 29 Serum concentration (ng/mL) of FP1 as an intact dimer over timefollowing a single SC administration in cynomolgus monkeys as determinedby immuno-affinity capture-LCMS analysis. Dimer Intact MS Data IA DataDay (Pooled Samples) (Average) 0.00 0 0 0.25 9625 8959 1.00 15799 142192.00 17671 17373 3.00 19130 17476 5.00 12284 15633 7.00 10808 1297110.00 8910 10143 14.00 5814 7034 18.00 4074 5054 22.00 2967 3694

The concentration of analytes in cynomolgus monkey serum after IV and SCadministration was also measured by immuno-affinity capture-trypsindigestion-LC-MS/MS analysis (Tables 30 and 31). Selected trypticpeptides, namely, ALV (ALVLIAFAQYLQQSPFEDHVK) (SEQ ID NO: 135), ASL(ASLEDLGWADWVLSPR) (SEQ ID NO: 136), and TDT (TDTGVSLQTYDDLLAK) (SEQ IDNO: 137), which are located within FP1 near the N-terminus of the HSAregion, the N-terminus of GDF15, and the C-terminal of GDF15,respectively. The peptides were monitored as surrogate peptides of FP1.The concentrations of all of the surrogate peptides were similar to eachother and the concentrations measured by immunoassay, demonstrating thatthe GDF15 sequence in FP1 remains intact and linked to the full HSAsequence in vivo.

TABLE 30 Serum concentration (ng/mL) of surrogate peptides representingvarious regions of FP1 over time following a single IV administration incynomolgus monkeys as determined by immuno-affinity capture- trypsindigestion-LC-MS/MS analysis. Time point FP1 Average (ng/mL) Std Dev IAData Day hour ALV TDT ASL ALV TDT ASL (ng/mL) 0.00 0 <LLOQ <LLOQ <LLOQN/A N/A N/A 0.0 0.04 1 32400.0 39766.7 33333.3 2623.0 3162.8 2722.734536.9 0.25 6 29100.0 27166.7 30600.0 3439.5 1006.6 2095.2 30328.1 1.0024 24366.7 23300.0 23800.0 4215.8 3996.2 4100.0 24088.7 2.00 48 19433.317733.3 18700.0 1457.2 1193.0 854.4 19448.6 3.00 72 18100.0 17200.017166.7 360.6 871.8 1001.7 17271.6 5.00 120 15966.7 14033.3 14233.31001.7 642.9 1011.6 16282.3 7.00 168 13733.3 11800.0 12100.0 1115.0600.0 300.0 13673.6 10.00 240 9303.3 8570.0 8570.0 2290.1 1682.2 1685.910973.0 14.00 336 5860.0 5890.0 6056.7 415.8 312.2 388.0 6878.9 18.00432 4143.3 4400.0 4226.7 374.3 52.9 571.2 4628.6 22.00 528 2830.0 3256.72753.3 355.4 420.0 319.0 3984.0

TABLE 31 Serum concentration (ng/mL) of surrogate peptides representingvarious regions of FP1 over time following a single SC administration incynomolgus monkeys as determined by immuno-affinity capture- trypsindigestion-LC-MS/MS analysis. Time point FPI Average (ng/mL) Std Dev IAData Day hour ALV TDT ASL ALV TDT ASL (ng/mL) 0.00 0 <LLOQ <LLOQ <LLOQN/A N/A N/A 0.0 0.25 6 9323.3 7430.0 8123.3 1900.3 2471.6 1954.1 8959.11.00 24 15233.3 13390.0 14533.3 2926.3 3222.0 2683.9 14219.2 2.00 4815366.7 14000.0 14166.7 2579.4 1646.2 2311.6 17373.0 3.00 72 17300.016033.3 15333.3 1571.6 1001.7 986.6 17476.0 5.00 120 15333.3 13366.713666.7 1222.0 1692.1 1501.1 15632.9 7.00 168 13333.3 11633.3 11700.0709.5 472.6 781.0 12970.9 10.00 240 8496.7 7376.7 8343.3 1475.5 189.01039.1 10143.2 14.00 336 6046.7 6116.7 6253.3 90.2 118.5 110.6 7034.518.00 432 4593.3 5250.0 4636.7 802.6 1157.5 621.7 5054.2 22.00 5283056.7 3490.0 3116.7 424.5 687.7 220.5 3693.7

Human plasma stability assay

The purpose of this study was to analyze the ex vivo stability of FP1 inhuman plasma. Fresh, non-frozen human plasma was generated fromheparinized blood from two subjects (one male and one female) bycentrifugation. FP1 was incubated in this matrix at 37° C. with gentlemixing, for 0, 4, 24 and 48 hours. The concentration of FP1 wasdetermined using an immunoassay method. The average percent differencefrom the starting concentration (0 hours) ranged from −4.1 to −12.9 anddid not increase over time, demonstrating that FP1 is stable in humanplasma for up to 48 hours ex vivo (Table 32 and FIG. 15 ).

TABLE 32 FP1 concentration (μg/mL) after 0, 4, 24, and 48 hours (hr) ofex vivo incubation in plasma obtained from two human subjects (Sub) asdetermined by immunoassay Sub 1 Sub 2 Male % Female % Average % Conc.Diff Conc. Diff Conc. Diff Ex Vivo Sample (μg/mL) T0 (μg/mL) T0 (μg/mL)T0 Plasma-T0 hr_FP1 11.503 N/A 12.649 N/A 12.076 N/A Plasma-T4 hr_FP110.524 −8.5 10.521 −16.8 10.523 −12.9 Plasma-T24 hr_ 9.934 −13.6 12.402−2.0 11.168 −7.5 FP1 Plasma-T48 hr_ 10.582 −8.0 12.575 −0.6 11.578 −4.1FP1

Immuno-affinity capture-LCMS was used to quantitate the concentration ofintact dimer present after incubation in human plasma. Concentrationsdetermined by this method were stable over time (0, 4, 24, and 48hours), demonstrating that FP1 remains an intact dimer in human plasmaex vivo up to 48 hours (Table 33 and FIG. 16 ).

TABLE 33 Average FP1 concentration (μg/mL) and % difference fromstarting concentration as an intact dimer after 0, 4, 24, and 48 hours(hr) of ex vivo incubation in plasma obtained from two human subjects asdetermined by immuno-affinity capture-LCMS analysis Dimer Conc. (ug/mL)% Difference  0 hr 15.8 100.0  4 hr 15.8 100.1 24 hr 15.9 100.9 48 hr15.2 96.0

Example 13: IR Strategy

Immune response (IR) assays will be developed for anti-drug antibody(ADA) detection in animals and clinical samples. The IR assay willidentify ADA-positive samples for comparison of ADA status withpharmacokinetic/toxicokinetic (PK/TK) results, enabling assessment ofFP1 exposure and pharmacokinetics. The clinical IR assay will be used toscreen serum samples, confirm specificity of ADA-positive samples, anddetermine the ADA titer for confirmed positive samples. Neutralizingantibody (NAb) assay development will follow for use in confirmedpositive samples from ADA-positive subjects in Phase 1 of the program.Additionally, determination of ADA cross-reactivity to endogenous GDF15will follow for use in Phase 2 of the program. An immunogenicity riskassessment will be conducted prior to the first in human (FIH) study,and additional immune response characterization assays can beimplemented if they are warranted.

Example 14: Toxicology Plan

Since the endogenous target receptor for GDF15 has not been identified,there is a lack of in vitro binding and functional data for FP1.However, single and multiple dose pharmacology and efficacy studies inrats, mice and cynomolgus monkeys have demonstrated activity of FP1 inthese species, showing its effect of reducing food intake, decreasingbody weight and modulating oral glucose tolerance. The rat and monkeywill be the rodent and non-rodent toxicology testing species,respectively, based on the efficacy results, with the understanding thatthe intrinsic potency of FP I on the receptor in these species (versusthat of humans) has not been fully characterized.

The Examples 15-19 involve characterization of another exemplary fusionprotein of the invention, described in Example 5, which has the aminoacid sequence of SEQ ID NO: 92 (encoded by nucleotide sequences SEQ IDNOs: 95 (codon optimization 1) or 110 (codon optimization 2)). Thisfusion protein is a fully recombinant protein that exists as a homodimerof a fusion of HSA (C34S) with the deletion variant of the mature humanGDF15 (201-308; SEQ ID NO: 8) through a 42-amino acid linker consistingof glycine and serine residues, GS-(GGGGS)8 (SEQ ID NO: 12). The singlenative free cysteine at position 34 of HSA has been mutated to serine.This particular HSA-GDF15 fusion protein will be referred to as “FP2” inthe following examples, for simplicity.

Example 15: Effects of FP2 on the Food Intake of C57B1/6 Mice

FP2 was evaluated for its ability to reduce food intake in male C57B1/6mice after a single dose. Male C57B1/6N mice (age 10-12 weeks) obtainedfrom Taconic Biosciences (Hudson, N.Y.) were used in the study. Micewere singly housed in a temperature-controlled room with 12-hourlight/dark cycle (6 am/6 pm) and allowed ad libitum access to water andchow. Male C57B1/6 mice were acclimated for a minimum of 72 hours in theBioDAQ cages; mice were then grouped based on food intake in the last 24hours into six groups of eight each. Between 4:00-5:00 pm, animals wereweighed and dosed with vehicle or compounds via subcutaneous injection.Change in food weight for each cage was recorded continuously by theBioDAQ system, for a period of 48 hours after compound administration.6xHis-FP1 was used as a comparator in this study.

FP2 had significant effects on reducing food intake at 12, 24 and 48hours after administration at all dose levels tested (Table 34). Therewas a reduction in percent change in food intake relative to PBS at alltime points and all dose levels (Table 35) in mice.

TABLE 34 Effect of a Single Dose of FP2 on Food Intake over 48 hours inC57Bl/6 Mice Cumulative Food Intake (g) Treatment 12 hours 24 hours 48hours PBS 3.7 ± 0.2 4.4 ± 0.2 8.8 ± 0.4 FP2, 1 nmol/kg 2.8 ± 0.4* 3.0 ±0.4** 6.9 ± 0.7* FP2, 4 nmol/kg 2.4 ± 0.2*** 3.1 ± 0.2** 7.0 ± 0.3* FP2,8 nmol/kg 1.9 ± 0.2**** 2.4 ± 0.1**** 6.2 ± 0.2*** FP2, 16 nmol/kg 1 8 ±0.1**** 2.7 ± 0.1*** 6.3 ± 0.4** 6xHis-FP1, 8 nmol/kg 2.3 ± 0.3*** 2.8 ±0.4** 6.6 ± 0.7** Data are expressed as Mean ± SEM. *p ≤ 0.05, versusPBS **p ≤ 0.01, versus PBS ***p ≤ 0.001, versus PBS ****p ≤ 0.0001,versus PBS, respectively Statistical analyses used: ANOVA and Dunnett'smultiple comparisons test. n = 8/group, except for 6xHis-FP1 8 nmol/kg(n = 6).

TABLE 35 Effect of a Single Dose of FP2 on Percent Reduction in FoodIntake (Relative to Vehicle) over 48 hours in C57Bl/6 Mice Percentage ofinhibition relative to PBS Treatment 12 hours 24 hours 48 hours PBS  0.0± 7.9  0.0 ± 7.7  0.0 ± 5.9 FP2, 1 nmol/kg 22.4 ± 12.5* 31.8 ± 10.5**21.8 ± 8.1* FP2, 4 nmol/kg 36.6 ± 7.2*** 30.4 ± 5.8** 20.2+4.7* FP2, 8nmol/kg 47.0 ± 6.2**** 45.7 ± 3.8**** 29 7-3 9*** FP2, 16 nmol/kg 49 2 ±4.1**** 38.1 ± 4.1*** 27.8 ± 5.1** 6 × His-FP1, 8 nmol/kg 36.9 ± 9.9***36.5 ± 8.8** 24.6 ± 8.2** The anorectic effect of FP2 is expressed asthe relative reduction in food intake compared with the respective PBScontrols. Data are expressed as Mean ± SEM. *p ≤ 0.05, versus PBS **p ≤0.01, versus PBS ***p ≤ 0.001, versus PBS ****p ≤ 0.0001, versus PBS,respectively Statistical analyses used: ANOVA and Dunnett's multiplecomparisons test. n = 8/group, except for 6 × His-FP1 8nmol/kg (n = 6).

Example 16: Effects of FP2 on Food Intake in Sprague Dawley Rats

FP2 was evaluated for its ability to reduce food intake and body weightgain in male Sprague-Dawley rats after a single dose. The animals wereobtained from Charles River (Wilmington, Mass.) at 200-225 g body weightand used within one week of delivery. They were housed one per cage onalpha dry bedding and a plastic tube for enrichment in atemperature-controlled room with 12-hour light/dark cycle. They wereallowed ad libitum access to water and were fed laboratory rodent diet;Irradiated Certified PicoLab® Rodent Diet 20, 5K75* (supplied fromPurina Mills, St. Louis, Mo. via ASAP Quakertown, Pa.). Animal weightswere taken and recorded for each rat prior to dosing.

Animals were acclimated for a minimum of 72 hours in the BioDAQ cages;rats were then grouped based on food intake in the last 24 hours intosix groups of eight each. Between 4:00-5:00 pm, animals were weighed anddosed with vehicle or compounds via subcutaneous injection. Change infood weight for each cage was recorded continuously by the BioDAQsystem, for a period of 48 hours after compound administration.6XHis-FP1 was used as a comparator in this study.

Dose-dependent reductions of food intake were tested after a single doseof FP2. No significant differences in food intake were observed at thedose of 0.3 nmol/kg. Significant effects in reduction of food intakewere observed 12 hours but not 24 or 48 hours at 1 nmol/kg. Significantreductions in food intake were observed at all time points for the 3 and10 nmol/kg dose levels (Table 36, FIG. 19 ). There was a reduction inpercent change in food intake relative to PBS at all time points and alldose levels (Table 37).

TABLE 36 Effect of a single dose of FP2 on food intake over 48 hours inSprague Dawley rats Cumulative Food Intake (g) Treatment 12 hours 24hours 48 hours PBS 21.7 ± 0.6 25.0 ± 0.8 53.1 ± 1.6 FP2, 0.3 nmol/Kg19.5 ± 0.9 22.9 ± 0.7 48.4 ± 1.5 FP2, 1 nmol/Kg 17.5 ± 0.9* 19.8 ± 0.847.0 ± 2.6 FP2, 3 nmol/Kg 16.1 ± 1.2** 17.0 ± 1.0** 39.2 ± 3.2** FP2, 10nmol/Kg 15.8 ± 0.9*** 16.5 ± 0.9** 36.5 ± 3.2** 6 × His-FP1 8 nmol/Kg15.0 ± 1.4*** 15.7 ± 1.2** 37.2 ± 4.5** Data are expressed as Mean ±SEM. *p ≤ 0.05, versus PBS **p ≤ 0.01, versus PBS ***p ≤ 0.001, versusPBS, respectively Statistical analyses used: ANOVA and Dunnett'smultiple comparisons test. n = 8/group

TABLE 37 Effect of a single dose of FP2 on percent reduction in foodintake (relative to vehicle)over 48 hours in Sprague Dawley rats.Percentage of inhibition relative to PBS Treatment 12 hours 24 hours 48hours PBS  0.0 ± 4.0  0.0 ± 4.6  0.0 ± 4.3 FP2 0.3 nmol/kg 10.0 ± 4.7 8.5 ± 4.1  8.8 ± 3.9 FP2 1 nmol/kg 19.3 ± 4.7* 20.8 ± 4.1 11.5 ± 5.5FP2 3 nmol/kg 25.9 ± 6.1** 32.0 ± 4.7** 26.2 ± 6.5** FP2 10 nmol/kg 27.4± 4.8*** 33 8 ± 4.1** 31.2 ± 6.4** 6 × His-FP1 8 nmol/kg 30.8 ± 6.6***37.0 ± 5.4** 29.9 ± 8.8** The anorectic effect of FP2 is expressed asthe relative reduction in food intake compared with the respective PBScontrols. Data are expressed as Mean ± SEM. *p ≤ 0.05, versus PBS **p ≤0.01, versus PBS ***p ≤ 0.001, versus PBS, versus PBS, respectivelyStatistical analyses used: ANOVA and Dunnett's multiple comparisonstest. n = 8/group

Example 17: Effects of FP2 on Food Intake, Body Weight and GlucoseHomeostasis in Diet-induced Obese (DIO) C57B1/6 Mice

FP2 was evaluated for its ability to reduce food intake and body weightand improve glucose homeostasis on repeat dosing in male DIO C57B1/6mice over a period of 8 days. Male DIO C57B1/6 mice (age 21 weeks, highfat-fed for 15 weeks) obtained from Taconic Biosciences (Hudson, NY)were used in the study. Mice were singly housed in atemperature-controlled room with 12-hour light/dark cycle (6 am/6 pm)and allowed ad libitum access to water and fed with Research Diet D12492(Research Diets, New Brunswick, N.J.). Mice were acclimated >1 week inthe mouse housing room prior to the experiment. The endpoints of thestudy were measurements of food intake, body weight, body compositionand glycemic endpoints (OGTT, blood glucose). One day prior to dosing,animals were weighed and grouped by body weight (BW). Mice were dosed bysubcutaneous injection. Animals dosed with FP2 received this compound onDay 0, Day 3, and Day 6, Day 9 and Day 12. The vehicle group androsiglitazone group received sterile PBS s.c. on these days as well.Rosliglitazone was provided in the diet at 0.015% w/w ad libitum. BW andfood intake were recorded daily, over a period of fifteen days. Bloodglucose was measured on Days 0, 7 and 13. An oral glucose tolerance test(OGTT) was performed on Day 14. Insulin levels were measured at selectedtime points during the OGTT. Mice were euthanized with CO₂ and terminalblood samples were collected for exposure via cardiac puncture on day15. A separate PK arm was run with three mice per dose group with atotal of 15 mice.

Exposure-response (E-R) Analysis for FP2 in DIO Mouse

Most animals in the pharmacodynamics (PD) (efficacy) arms hadundetectable drug concentrations on the last study day when thepharmacokinetics (PK) samples were obtained, potentially due toimmunogenicity. Therefore, the mean PK profiles from the PK arms,instead of individual PK from the PD arms, were used to conductexposure-response (from day 3, 6 and 9, respectively) for the % weightchange from baseline in the PD arms at the corresponding dose level.This method assumes that the PK arms behave similarly to the PD arms interms of drug exposure.

The E_(max) model (GraphPad Prism 6, log(agonist) vs. response) was usedto correlate exposure with response data (log transformed drugconcentrations). Hill Slope was set to be 1. Note that the model fittedEC₁₀ to EC₅₀ values were within two fold amongst day 3, 6 and 9, despitethat the E_(max) estimates were different (E_(max)=−4.26%, −8.18% and−9.85%, respectively). Some animals on day 9 also showed the loss ofdrug exposure, due to potential ADA formation and therefore, the E-Rparameter estimates based on day 9 data should be interpreted withcaution.

The effects of two weeks of exposure of FP2 on food intake, body weight,glucose homeostasis, and liver fat content was assessed in diet inducedobese male C57B1/6 mice. Trough exposure between 1.7 and 3.3 nM FP2 forthe 0.3 nmol/kg treatment group, between 7.1 and 14 nM for the 1.0nmol/kg treatment group, between 20.8 and 41.6 nM for the 3.0 nmol/kgtreatment group, and between 28.5 and 112.9 nM FP2 for the 10 nmol/kgtreatment group was maintained until day 9 in the PK arm of the study(n=2 or 3, Table 49). After day 9, a decrease in circulating levels wasobserved in the majority of animals despite continued q3d dosing (Table49). Consistent with this accelerated clearance, the majority of animalsin the PD arm of the study had undetectable circulating levels of FP2 onday 15 (Table 50).

Treatment of DIO mice with FP2 q3d reduced food intake (Table 38), bodyweight (Table 39, 40 and FIG. 20 ) and fed blood glucose compared tovehicle treatment (Table 43 and FIG. 23 ). A significant reduction infood intake was seen on day 2, day 5, and day 8 for 0.3 nmol/kg, fromday 1 through day 7 for 1.0 nmol/kg, on day 1, day 2, day 4 through day6, and day 8 for 3.0 nmol/kg, and on day 1, day 3 through day 6, day 8and day 9 for 10.0 nmol/kg. Percent body weight changes were significantfrom day 5 through day 13 for 0.3 nmol/kg, from day 3 through day 13 for1.0 nmol/kg and 10.0 nmol/kg, and from day 4 through day 13 for 3.0nmol/kg. Changes in grams of body weight were significant from day 8 for0.3 nmol/kg, from day 6 for 1.0 nmol/kg, from day 7 for 3.0 nmol/kg andfrom day 5 for 10.0 nmol/kg. Decreases in fed blood glucose levels weresignificant on day 7 for the animals in the 3.0 nmol/kg dose level andwere significant on day 13 for the animals in the 3.0 and 10.0 nmol/kgdose levels.

DIO mice treated with FP2 q3d had improved glucose tolerance on day 14compared to vehicle treatment during an oral glucose challenge (Table41; FIG. 21A and 21B). Glucose was significantly lower at 30 minutes forthe 0.3 nmol/kg group, at 60 minutes and 120 minutes for the 1.0 nmol/kggroup, at 120 minutes for the 3.0 nmol/kg group, and at 30, 90, and 120minutes for the 10.0 nmol/kg group. Total area under the curve wassignificant for all dose groups. Insulin levels during the glucosechallenge were significantly lower for 0.3 and 10.0 nmol/kg groups at 30minutes (Table 42; FIGS. 22A and 22B). In addition, compared to vehicletreated animals, there was a significant reduction in the calculatedfasted HOMA-IR in DIO mice after 14 days of treatment with FP2 q3d at10.0 nmol/kg indicative of improved insulin sensitivity (Table 44 andFIG. 24 ).

Body composition was measured by MM on day -1 before the start of thestudy and on day 13 (Table 47 and Table 48). DIO mice treated with FP2at 1.0 nmol/kg and 10.0 nmol/kg had significant reductions in fat masson day 13; whereas there were no changes in lean mass for any treatmentgroups. On day 13, the 10.0 nmol/kg treatment group had a significantincrease in percent lean mass and a significant reduction in percent fatmass compared to the vehicle treated group. Changes from day -1 to day13 were significant for lean mass in the 0.3 nmol/kg, 1.0 nmol/kg, and10.0 nmol/kg treatment groups and were significant for percent lean massin the 1.0, 3.0, and 10.0 nmol/kg treatment groups. Changes from day -1to day 13 were significant for fat mass and percent lean mass in alltreatment groups compared to vehicle.

There was no significant difference in endogenous mouse GDF15 serumlevels between vehicle treated animals and mice treated with FP2 q3d for15 days (Table 46).

Conclusion: the results suggest that the higher drug exposure isgenerally associated with greater % weight change from baseline on apopulation level across the studied dose groups on day 3, 6 and 9.

Exposure to FP2 over two weeks led to reduced food intake, decreasedbody weight, decreased blood glucose, improved glucose tolerance andinsulin sensitivity in DIO mice. Significant decreases in food intakeover multiple days were achieved at 1.0, 3.0, and 10.0 nmol/kg q3d. Bodyweight was decreased significantly starting three to five days after theinitiation of the study. Fed blood glucose on day 13 was significantlydecreased after q3d administration of FP2 at 3.0 and 10.0 nmol/kg.Insulin sensitivity represented by significantly decreased fastingHOMA-IR was achieved 14 days after 10.0 nmol/kg FP2 administered q3d. Onday 13, a significant increase in percent lean mass and a significantreduction in percent fat mass was observed in DIO mice treated q3d with10.0 nmol/kg FP2.

TABLE 38 Effect of FP2 on daily food intake (g) over 13 days oftreatment. Treatment Vehicle FP2 (nmol/kg) Rosiglitazone Day N/A 0.3 1.03.0 10.0 10 mpk/day 0 2.0 ± 0.1 1.9 ± 0.1 2.2 ± 0.2 1.9 ± 0.2 1.9 ± 0.22.2 ± 0.1 1 2.4 ± 0.2 2.1 ± 0.1 1.6 ± 0.1* 1.5 ± 0.1* 1.3 ± 0.1* 2.7 ±0.1 2 2.5 ± 0.1 1.8 ± 0.1* 1.7 ± 0.2* 1.9 ± 0.1* 2.0 ± 0.1 3.0 ± 0.2 32.5 ± 0.1 2.1 ± 0.1 1.7 ± 0.1* 1.9 ± 0.1 1.8 ± 0.1* 2.8 ± 0.2 4 2.6 ±0.1 2.0 ± 0.1 1.8 ± 0.1* 1.9 ± 0.1* 1.9 ± 0.1* 2.9 ± 0.2 5 2.8 ± 0.1 2.1± 0.2* 2.2 ± 0.1* 2.2 ± 0.0* 1.9 ± 0.1* 2.7 ± 0.2 6 2.8 ± 0.1 2.3 ± 0.22.1 ± 0.1* 2.2 ± 0.1* 2.0 ± 0.1* 2.8 ± 0.2 7 2.6 ± 0.2 2.3 ± 0.1 2.0 ±0.1* 2.0 ± 0.2 2.1 ± 0.1 2.9 ± 0.2 8 2.7 ± 0.2 2.1 ± 0.1* 2.2 ± 0.1 2.0± 0.1* 2.0 ± 0.1* 3.1 ± 0.2 9 2.8 ± 0.1 2.3 ± 0.1 2.3 ± 0.2 2.3 ±0.2{circumflex over ( )} 2.1 ± 0.2* 3.2 ± 0.1 10 2.6 ± 0.1 2.5 ± 0.1 2.4± 0.2 2.3 ± 0.2{circumflex over ( )} 2.1 ± 0.2 3.0 ± 0.2 11 2.9 ± 0.12.5 ± 0.1 2.8 ± 0.2 2.6 ± 0.4 2.6 ± 0.1 3.3 ± 0.1 12 2.8 ± 0.1 2.7 ± 0.22.8 ± 0.1 2.8 ± 0.2 2.9 ± 0.1 3.1 ± 0.2 13 2.7 ± 0.1 2.5 ± 0.2 2.6 ± 0.12.6 ± 0.1 2.2 ± 0.1 3.1 ± 0.2 Values represent mean ± SEM or data from 8animals per time per group, except n = 7 when noted by {circumflex over( )} *p < 0.05, versus vehicle Statistical analyses used: Two-Way ANOVARM, Tukey's multiple comparison test

TABLE 39 Effect of FP2 on Percent Body Weight Change Over 13 days ofTreatment Treatment Vehicle FP2 (nmol/kg) Rosiglitazone Day N/A 0.3 1.03.0 10.0 10 mpk/day −1 −0.1 ± 0.4   0.5 ± 0.2 −0.1 ± 0.1 −0.4 ± 0.2   0.0 ± 0.6 0.1 ± 0.2 0   0.0 ± 0.0   0.0 ± 0.0   0.0 ± 0.0   0.0 ± 0.0   0.0 ± 0.0 0.0 ± 0.0 1 −0.4 ± 0.5 −1.0 ± 0.3 −2.2 ± 0.4 −1.8 ± 0.6 −2.4 ± 0.5 1.3 ± 0.4 2 −0.4 ± 0.3 −2.2 ± 0.4 −3.4 ± 0.6 −3.0 ± 0.6 −3.1 ± 0.4 1.3 ± 0.5 3 −0.3 ± 0.4 −2.3 ± 0.4 −4.2 ± 0.6* −3.5 ± 0.6 −4.3 ± 0.5* 1.9 ± 0.5 4 −0.3 ± 0.5 −2.9 ± 0.5 −5.3 ± 0.6* −4.9 ± 0.7* −5.8 ± 0.7* 1.8 ± 0.7 5 −0.2 ± 0.4 −4.2 ± 0.7* −6.2 ± 0.6* −5.6 ± 0.7* −6.8 ± 0.7* 1.7 ± 0.8 6 −0.4 ± 0.6 −5.1 ± 0.7* −7.4 ± 0.8* −6.9 ± 0.7* −8.5 ± 0.9* 1.1 ± 0.9 7   0.2 ± 0.8 −5.1 ± 0.7* −7.7 ± 0.9* −7.4 ± 0.7* −8.7 ± 0.9* 2.0 ± 1.0 8   0.2 ± 1.0 −6.1 ± 0.8* −7.9 ± 0.9* −8.0 ± 0.8* −9.7 ± 0.8* 2.4 ± 1.1 9   0.5 ± 1.1 −6.0 ± 0.9* −8.4 ± 0.9* −8.8 ± 0.9*−10.1 ± 1.0* 3.1 ± 1.0 10   1.1 ± 1.2 −5.7 ± 0.8* −8.1 ± 0.9* −8.9 ±0.9* −10.7 ± 1.2* 3.5 ± 1.2 11   1.2 ± 1.3 −6.1 ± 0.9* −8.2 ± 0.8* −8.6± 1.3* −11.1 ± 1.4* 3.7 ± 1.2 12   1.4 ± 1.3 −6.3 ± 1.2* −7.7 ± 0.8*−8.2 ± 1.4* −10.9 ± 1.4* 4.1 ± 1.3 13   1.7 ± 0.9 −5.8 ± 1.4* −7.1 ±1.0* −7.2 ± 1.4* −10.9 ± 1.4* 4.7 ± 1.3 Values represent mean ± SEM fordata from 8 animals per time per group *p < 0.05, versus VehicleStatistical analyses used: Two-Way ANOVA RM, Tukey's multiple comparisontest

TABLE 40 Effect of FP2 on body weight change (g) over 13 days oftreatment Treatment Vehicle FP2 (nmol/kg) Rosiglitazone Day N/A 0.3 1.03.0 10.0 10 mpk/day −1 44.6 ± 0.6 44.5 ± 0.6 44.5 ± 0.6 44.5 ± 0.6 44.5± 0.6 44.5 ± 0.6 0 44.6 ± 0.6 44.3 ± 0.6 44.6 ± 0.6 44.7 ± 0.6 44.6 ±0.7 44.4 ± 0.6 1 44.5 ± 0.7 43.8 ± 0.6 43.6 ± 0.6 43.9 ± 0.7 43.5 ± 0.745.0 ± 0.7 2 44.5 ± 0.7 43.3 ± 0.7 43.1 ± 0.6 43.3 ± 0.7 43.2 ± 0.7 45.0± 0.7 3 44.5 ± 0.7 43.2 ± 0.7 42.7 ± 0.6 43.1 ± 0.7 42.6 ± 0.6 45.3 ±0.7 4 44.5 ± 0.7 43.0 ± 0.7 42.2 ± 0.6 42.5 ± 0.7 42.0 ± 0.7 45.2 ± 0.75 44.5 ± 0.7 42.4 ± 0.7 41.8 ± 0.5 42.2 ± 0.7 41.5 ± 0.6* 45.2 ± 0.7 644.5 ± 0.7 42.0 ± 0.7 41.3 ± 0.6* 41.6 ± 0.7 40.7 ± 0.5* 44.9 ± 0.8 744.7 ± 0.7 42.0 ± 0.7 41.1 ± 0.6* 41.4 ± 0.7* 40.6 ± 0.5* 45.3 ± 0.8 844.7 ± 0.7 41.6 ± 0.7* 41.1 ± 0.7* 41.1 ± 0.7* 40.2 ± 0.6* 45.5 ± 0.8 944.8 ± 0.7 41.6 ± 0.8* 40.8 ± 0.7* 40.8 ± 0.8* 40.0 ± 0.8* 45.8 ± 0.7 1045.1 ± 0.7 41.8 ± 0.8* 41.0 ± 0.8* 40.8 ± 0.8* 39.8 ± 0.8* 46.0 ± 0.8 1145.2 ± 0.8 41.6 ± 0.8* 40.9 ± 0.7* 40.8 ± 0.9* 39.6 ± 0.9* 46.1 ± 0.8 1245.3 ± 0.7 41.5 ± 0.9* 41.2 ± 0.7* 41.0 ± 0.9* 39.7 ± 0.9* 46.3 ± 0.9 1345.4 ± 0.7 41.7 ± 0.9* 41.4 ± 0.8* 41.5 ± 0.9* 39.7 ± 0.9* 46.5 ± 0.9Values represent mean ± SEM for data from 8 animals per time per group*p < 0.05, versus Vehicle Statistical analyses: Two-Way ANOVA RM,Tukey's multiple comparison test

TABLE 41 Effect of FP2 on blood glucose (mg/dL) levels during an OGTTafter 14 days of treatment Dose Total AUC Δ AUC (nmol/ Time afterGlucose Challenge (min) 120 min) 120 min) Treatment kg) 0 30 60 90 120(mg/dL/ (mg/dL/ Vehicle NA 161 ± 10 225 ± 17 228 ± 14 208 ± 18 213 ± 1925429 ± 1228 6094 ± 1430 FP2 0.3 144 ± 4 163 ± 14* 209 ± 11 180 ± 12 171± 7 21270 ± 399* 3985 ± 521  1.0 151 ± 9 179 ± 18 188 ± 7 179 ± 13 163 ±8* 21096 ± 754* 2972 ± 907  3.0 149 ± 8 180 ± 18 179 ± 11* 164 ± 6 163 ±9* 20338 ± 876* 2426 ± 1058 10.0 134 ± 6 163 ± 7* 190 ± 13 152 ± 7* 163± 8* 19599 ± 614* 3539 ± 1198 Rosiglitazone 10 mpk/ 132 ± 9 152 ± 10*162 ± 13* 138 ± 9* 159 ± 9* 17904 ± 632* 2139 ± 1179 day Valuesrepresent mean ± SEM for data from 8 animals per time per group *p <0.05, versus Vehicle Statistical analyses: Two-Way ANOVA RM, Tukey'smultiple comparison test for Glucose Values; One-Way ANOVA, Tukey'smultiple comparison test for AUC

TABLE 42 Effect of FP2 on insulin (ng/mL) levels during an OGTT after 14days of treatment Dose Time after Glucose Total AUC (nmol/ Challenge(min) (ng/mL/ Treatment kg) 0 30 90 90 min) Vehicle NA 4.2 ± 0.7 12.2 ±2.3 3.6 ± 0.5 716.1 ± 122.0 FP2  0.3 2.9 ± 0.6  5.8 ± 1.8* 2.4 ± 0.4377.1 ± 97.3  1.0 2.9 ± 0.4  8.2 ± 2.6 2.6 ± 0.4 491.8 ± 118.2  3.0 3.0± 0.3  8.4 ± 1.5 2.5 ± 0.3 498.6 ± 77.0 10.0 2.4 ± 0.4  4.3 ± 0.5* 2.1 ±0.4 295.5 ± 32.0* Rosiglita- 10 mpk/ 0.9 ± 0.1  1.8 ± 0.2* 0.8 ± 0.1118.5 ± 9.6* zone day Values represent mean ± SEM for data from 8animals per time per group *—p < 0.05, versus Vehicle Statisticalanalyses: Two-Way ANOVA RM, Tukey's multiple comparison test for insulinValues; One-Way ANOVA, Tukey's multiple comparison test for AUC

TABLE 43 Effect of FP2 on fed blood glucose (mg/dL) levels Time afterstart of treatment Dose (days) Treatment (nmol/kg) 0 7 13 Vehicle NA 162± 5 159 ± 6 180 ± 9 FP2  0.3 143 ± 5 150 ± 9 168 ± 9  1.0 153 ± 12 135 ±7 159 ± 7  3.0 148 ± 3 127 ± 6* 151 ± 8* 10.0 146 ± 6 136 ± 4 148 ± 3*Rosiglitazone 10 mpk/day 137 ± 7 117 ± 4* 134 ± 6* Values represent mean± SEM for data from 8 animals per time per group *—p < 0.05, versusVehicle Statistical analyses: Two-Way ANOVA RM, Tukey's multiplecomparison test

TABLE 44 Fasted HOMA-IR in DIO mice after 14 days of q3d treatement withFP2 Dose HOMA- Treatment (nmol/kg) IR Vehicle NA 48.7 ± 9.2 FP2  0.130.1 ± 6.6  1.0 30.5 ± 3.8  3.0 31.3 ± 3.2 10.0 22.7 ± 3.1*Rosiglitazone 10 mpk/day  8.7 ± 1.0* Values represent mean ± SEM fordata from 8 animals per time per group *—p < 0.05, versus VehicleStatistical analyses: One-Way ANOVA, Tukey's multiple comparison test

TABLE 45 Liver weight after 15 days of treatment with FP2 q3d in DIOmice Liver Dose Liver Weight (% Treatment (nmol/kg) Weight, (g) of body)Vehicle NA 1.9 ± 0.1 4.3 ± 0.3 FP2  0.1 1.7 ± 0.1 4.0 ± 0.1  1.0 1.7 ±0.0 4.2 ± 0.1  3.0 1.8 ± 0.1 4.2 ± 0.1 10.0 1.7 ± 0.1 4.2 ± 0.1Rosiglitazone 10 mpk/day 1.9 ± 0.1 4.1 ± 0.2 Values represent mean ± SEMor data from 8 animals per time per group *—p < 0.05, versus VehicleStatistical analyses: One-Way ANOVA, Tukey's multiple comparison test

TABLE 46 Serum mouse GDF15 (pg/mL) levels after 15 days of treatementwith FP2 q3d in DIO mice Dose Treatment (nmol/kg) mGDF15 Vehicle NA258.3 ± 21.4 FP2  0.1 214.1 ± 10.3  1.0 191.6 ± 12.7  3.0 254.5 ± 28.610.0 202.0 ± 10.0 Rosiglitazone 10 mpk/day 509.6 ± 26.2* Valuesrepresent mean ± SEM or data from 8 animals per time per group *—p <0.05, versus Vehicle Statistical analyses: One-Way ANOVA, Tukey'smultiple comparison test

TABLE 47 Effect of JNJ-64739090 q3d in DIO mice on body composition (g)measured by MRI Dose Treat- (nmol/ Fat (g) Fat (g) Lean (g) Lean (g) ΔLean ment kg) Day −1 Day 13 Δ Fat (g) Day −1 Day 13 (g) Vehicle NA 17.4± 0.6 18.2 ± 0.6   0.9 ± 0.3 25.4 ± 0.3 25.1 ± 0.3 −0.4 ± 0.2 FP2 0.317.0 ± 0.6 15.2 ± 0.8 −1.9 ± 0.6* 25.8 ± 0.7 24.5 ± 0.5 −1.2 ± 0.2* 1.017.0 ± 0.6 14.9 ± 0.6* −2.1 ± 0.4* 25.8 ± 0.5 24.6 ± 0.4 −1.2 ± 0.2* 3.017.4 ± 0.7 15.1 ± 1.1 −2.3 ± 0.5* 25.3 ± 0.4 24.2 ± 0.4 −1.1 ± 0.1 10.016.7 ± 0.6 13.2 ± 0.8* −3.6 ± 0.5* 26.1 ± 0.6 24.5 ± 0.6 −1.7 ± 0.2*Rosi- 10 mpk/ 17.2 ± 0.6 19.1 ± 0.5   1.9 ± 0.6 25.5 ± 0.7 25.1 ± 0.6−0.4 ± 0.2 glitazone day Values represent mean ± SEM for data from 8animals per time per group *p < 0.05, versus Vehicle Statisticalanalyses: One-Way ANOVA, Tukey's multiple comparison test

TABLE 48 Effect of FP2 q3d in DIO mice on body composition (%) measuredby MRI Dose Treat- (nmol/ Fat (%) Fat (%) Lean (%) Lean (%) Δ Lean mentkg) Day −1 Day 13 Δ Fat (%) Day −1 Day 13 (%) Vehicle NA 38.1 ± 1.0 40.1± 1.0   2.1 ± 0.3 55.9 ± 0.9 55.3 ± 0.9 −0.6 ± 0.3 FP2 0.3 37.6 ± 1.236.3 ± 1.3 −1.3 ± 0.9* 56.8 ± 1.3 59.0 ± 1.4   2.2 ± 0.8 1.0 37.2 ± 1.135.8 ± 0.9 −1.4 ± 0.5* 56.7 ± 1.1 59.5 ± 1.0   2.8 ± 0.6* 3.0 38.3 ± 1.436.1 ± 2.0 −2.3 ± 0.7* 55.9 ± 1.2 58.7 ± 2.0   2.8 ± 0.8* 10.0 36.9 ±1.1 33.1 ± 1.5* −3.8 ± 0.8* 57.5 ± 1.0 61.7 ± 1.4*   4.2 ± 0.7* Rosi- 10mpk/ 38.0 ± 1.2 41.1 ± 0.9   3.1 ± 0.9 56.2 ± 1.2 53.9 ± 0.8 −2.3 ± 0.8glitazone day Values represent mean ± SEM for data from 8 animals pertime per group *p < 0.05, versus Vehicle Statistical analyses: One-WayANOVA, Tukey's multiple comparison test

TABLE 49 FP2 serum exposures (nM) of the PK arm during q3d treatment inDIO mice Time Post Initial Dose (Time post last dose) Treatment SubjectDay 14 Day 15 Day 17 Group ID Day 3 Day 6 Day 9 Day 12 (48 hr) (72 hr)(120 hr)  0.1 9 1.930 2.993 <LOQ <LOQ <LOQ <LOQ <LOQ nmol/ 10 1.8533.369 2.817 <LOQ <LOQ <LOQ <LOQ kg 11 1.715 2.637 2.568 2.709 3.4411.313 <LOQ  1.0 9 8.198 9.660 9.645 <LOQ <LOQ <LOQ <LOQ nmol/ 10 7.07310.595 8.966 <LOQ <LOQ <LOQ <LOQ kg 11 6.689 13.967 10.802 <LOQ <LOQ<LOQ <LOQ  3.0 9 20.802 26.329 27.863 <LOQ <LOQ <LOQ <LOQ nmol/ 1023.563 32.020 41.576 1.168 <LOQ <LOQ <LOQ kg 11 21.704 30.101 <LOQ <LOQ<LOQ <LOQ <LOQ 10.0 9 28.495 33.050 <LOQ <LOQ <LOQ <LOQ <LOQ nmol/ 1070.779 112.898 <LOQ <LOQ <LOQ <LOQ <LOQ kg 11 70.404 111.767 55.117 <LOQ<LOQ <LOQ <LOQ Data are expressed as concentration for each animal. <LOQ= below limit of quantitation; LOQ is 0.494 nM ** Values at Day 3, 6, 9and 12 are immediately prior to next dose

TABLE 50 Terminal serum exposures (nM) of FP2 after 15 days of q3dtreatment in DIO mice Treatment Group (nmol/kg) Animal ID 0.1 1.0 3.010.0 1 2.567 <LOQ <LOQ <LOQ 2 <LOQ <LOQ <LOQ 44.149 3 <LOQ <LOQ <LOQ<LOQ 4 1.144 0.678 <LOQ <LOQ 5 <LOQ <LOQ <LOQ <LOQ 6 3.440 <LOQ <LOQ<LOQ 7 2.727 <LOQ <LOQ <LOQ 8 <LOQ <LOQ <LOQ <LOQ Data are expressed asconcentration for each animal. <LOQ = below limit of quantitation; LOQis 0.494 nM

Example 18: FP2 Multispecies Pharmacokinetics and Immune Response

Mouse pharmacokinetics

The pharmacokinetic properties of FP2 were evaluated when administeredsubcutaneously to female C57B1/6 mice. FP2 was administeredsubcutaneously (n=5 samples per time point) and intravenously (n=5samples per time point) to female C57B1/6 mice (Sage Laboratories, StLouis, Mo.) at a dose level of 2.0 mg/kg in PBS, (pH 7.3-7.5). Thecollection of sample at the last time point was via a terminal bleed.Blood samples were collected, serum processed and drug concentrationswere measured up to 168 hours. The levels of FP2 were measured using animmunoassay method. The drug concentration profiles in plasma aresummarized in Table 51 and 52 and illustrated in FIG. 25 .

Pharmacokinetic analysis of FP2 in C57B1/6 mice demonstrated a terminalhalf-life of ˜1.51 and ˜1.76 days following IV and SC dosingrespectively, with a mean bioavailability of ˜61% following SCadministration.

TABLE 51 Serum concentration (ng/mL) of FP2 following a singlesubcutaneous (SC) dose in C57Bl/6 mice FP2-SC Dose Animal Animal AnimalAnimal Animal 31 32 33 35 36 Average Result Result Result Result ResultResult Std Dev Timepoint (ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL)(ng/mL)   4 hr 11310.11 6323.44 23489.21 4357.74 4835.54 10063.21 7995.0 24 hr 12378.58 10896.80 17769.03 15928.14 15404.08 14475.33 2785.0  72hr 5127.95 5569.27 6727.37 7909.66 7550.32 6576.91 1210.5  96 hr 3059.833350.25 4042.04 4095.15 4431.94 3795.84 569.0 168 hr 784.61 1202.661364.68 1557.60 1678.02 1317.51 348.9

TABLE 52 Serum concentration (ng/mL) of FP2 following a singleintravenous (IV) dose in C57Bl/6 mice FP2-IV Dose Animal Animal AnimalAnimal Animal 37 40 41 45 48 Average Result Result Result Result ResultResult Std Dev Timepoint (ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL)(ng/mL)   1 hr 49573.59 42382.59 40001.18 43085.75 46443.59 44297.343742.9  24 hr 22173.85 19120.39 19393.78 19798.29 17459.96 19589.251696.7  72 hr 8334.33 7989.10 8573.44 9086.37 8186.68 8433.98 422.5  96hr 4478.83 4699.80 4749.93 5331.39 4579.08 4767.81 332.3 168 hr 1044.241419.64 1393.44 1835.63 979.49 1334.49 343.6

TABLE 53 Pharmacokinetic parameters of FP2 following 2 mg/kg IV and 2mg/kg SC administration in C57B1/6 mice. CL or Vz or AUC_(0-last)AUC_(0-inf) t_(1/2) CL/F Vz/F C_(max) T_(max)* (day* (day* Route (day)(ml/day/kg) (ml/kg) (ng/ml) (day) ng/ml) ng/ml) SC Mean 1.76 43 10815619 1 44972 48379 SD 0.16 8 20 4870 8563 9147 IV Mean 1.51 25 55 442970.042 76269 79253 SD 0.18 1 6 3743 3788 4051

Rat Pharmacokinetics

FP2 was administered subcutaneously (n=5 samples per time point) andintravenously (n=5 samples per time point) to female Sprague-Dawley rats(Sage Laboratories, St. Louis, MO) at a dose level of 2.0 mg/kg in PBS,(pH 7.3-7.5). The collection of sample at the last time point was via aterminal bleed. Blood samples were collected, serum processed and drugconcentrations were measured up to 168 hours. The levels of FP2 weremeasured using an immunoassay method. The drug concentration profiles inplasma are summarized in Table 54 and 55, and illustrated in FIG. 26 .Pharmacokinetic parameters calculated from these data are summarized inTable 56.

Pharmacokinetic analysis of FP2 in Sprague Dawley rats demonstrated aterminal half-life of ˜1.46 and ˜1.37 days following IV and SC dosingrespectively, with a mean bioavailability of ˜28% following SCadministration.

TABLE 54 Serum concentration (ng/mL) of FP2 following a singlesubcutaneous (SC) dose in Sprague-Dawley rats. FP2-SC Dose Animal AnimalAnimal Animal Animal 01 02 03 04 05 Average Std Result Result ResultResult Result Result Dev Timepoint (ng/ml) (ng/ml) (ng/ml) (ng/ml)(ng/ml) (ng/ml) (ng/ml)   4 hr 730.95 257.94 469.95 496.34 400.65 471.16172.2  24 hr 9236.27 6658.79 7728.92 7449.56 6502.60 7515.23 1092.1  72hr 4559.04 4464.72 5619.82 4287.25 3844.15 4555.00 655.6  96 hr 2791.972452.35 3387.55 2316.06 2176.00 2624.79 483.8 168 hr 622.32 606.36<80.00 514.19 476.49 554.84 70.7

TABLE 55 Serum concentration (ng/mL) of FP2 following a singleintravenous (IV) dose in Sprague-Dawley rats. FP2-IV Dose Animal AnimalAnimal Animal Animal 06 07 08 09 10 Average Std Result Result ResultResult Result Result Dev Timepoint (ng/ml) (ng/ml) (ng/ml) (ng/ml)(ng/ml) (ng/ml) (ng/ml)   1 hr 60785.80 47392.80 43046.54 46014.2544547.65 48357.41 7134.5  24 hr 25470.36 24729.88 21157.29 20497.3221459.71 22662.91 2267.1  72 hr 8208.46 9262.13 8866.76 8635.93 8843.838763.42 384.1  96 hr 4433.18 4833.22 4995.63 4630.60 4604.13 4699.35218.1 168 hr 1083.14 1469.35 1614.61 1394.17 1053.89 1323.03 245.7

TABLE 56 Pharmacokinetic parameters of FP2 following 2 mg/kg IV and 2mg/kg SC administration in Sprague-Dawley Rats. CL or Vz or AUC_(0-last)AUC_(0-inf) t_(1/2) CL/F Vz/F C_(max) T_(max)* (day* (day* Route (day)(ml/day/kg) (ml/kg) (ng/ml) (day) ng/ml) ng/ml) SC Mean 1.37 84 165 75151 22614 24036 SD 0.04 10 17 1092 2509 2893 IV Mean 1.46 23 49 483570.042 83271 86089 SD 0.12 2 6 7135 6081 5820

Monkey Pharmacokinetics

FP2 was administered subcutaneously at 1 mg/kg and intravenously at 1mg/kg to three male cynomolgus monkeys each in PBS, (pH 7.0-7.6). Bloodsamples were collected, plasma processed and drug concentrations weremeasured up to 21 days.

The pharmacokinetics (PK) of FP2 was characterized followingadministration of a single dose IV (1.0 mg/kg) and SC (1.0 mg/kg) incynomolgus monkeys. The plasma drug concentration-time profile after SCadministration is summarized in Tables 57 and 58 for immunoassay andLCMS analyses respectively and after IV administration in Tables 59 and60 for immunoassay and LCMS analyses respectively. The immunoassay datais graphed in FIG. 27 , and the LCMS data is represented in FIG. 28 .

Using results from the immunoassay analysis, the mean NCA-based terminalhalf-life (t1/2) for FP2 was ˜7.05 and ˜8.51 days following IV and SCdosing, respectively. The mean PK parameters following IV and SCadministration are summarized in Table 61. Using results from theimmunoassay bioanalysis, the mean non-compartment model estimatedterminal half-life (t1/2) for FP2 was 7.05 and 8.51 days following IVand SC dosing, respectively. The mean bioavailability (F%) of FP2 wasestimated to be ˜98.5% based on AUC_(0-last) and estimated to be ˜109.2%based on AUC_(0-Inf) in cynomolgus monkeys following SC administration.

TABLE 57 Plasma concentration (ng/mL) of FP2 measured by immunoassayfollowing a single SC dose in cynomolgus monkeys. Immunoassay AnimalAnimal Animal Time (hr) 704 705 706 SC Ave Std Dev  0 <80.0 <80.0 <80.0<80.0 N/A  6 7365.3 6128.5 6056.9 6516.9 735.6  24 19716.8 10903.810554.7 13725.1 5191.9  48 18191.7 14353.7 14464.7 15670.0 2184.5  7217813.9 14207.5 12684.0 14901.8 2634.5 120 13823.9 11445.8 10590.811953.5 1675.3 168 12359.6 10103.8 9467.8 10643.8 1519.7 240 9457.97642.6 8109.1 8403.2 942.7 336 6796.8 5679.8 5235.7 5904.1 804.3 4325581.3 3830.6 3746.5 4386.1 1035.9 528 4126.2 2613.4 2879.3 3206.3 807.7N/A = not applicable

TABLE 58 Plasma concentration (ng/mL) of FP2 measured by LCMS followinga single SC dose in cynomolgus monkeys. LC/MS Animal Animal Animal Time(hr) 704 705 706 SC Ave SEM  0 <1000 <1000 <1000 <1000 N/A  6 6110.05910 6200.0 6073.3 85.7  24 # 10420.0 11300.0 10860.0 359.3  48 16560.013510.0 14960.0 15010.0 880.8  72 13690.0 13450.0 13380.0 13506.7 93.9120 11040.0 # # 11040.0 N/A 168 # 8680.0 9400.0 9040.0 293.9 240 6940.07070.0 7140.0 7050.0 58.6 336 4400.0 4580.0 4430.0 4470.0 55.7 432 —2910.0 — 2910.0 N/A 528 <1000 <1000 <1000 <1000 N/A — = initial runfailed, not enough sample for repeat analysis # = mislabeled tube,sample excluded from the analysis N/A = not applicable

TABLE 59 Plasma concentration (ng/mL) of FP2 measured by immunoassayfollowing a single IV dose in cynomolgus monkeys. Immunoassay Time (hr)Animal 701 Animal 702 Animal 703 IV Ave Std Dev 0 <80.0 <80.0 <80.0<80.0 N/A 1 29938.3 31139.0 23545.3 28207.6 4082.0 6 24711.9 21173.020434.4 22106.4 2286.5 24 19648.4 21420.9 8662.7 16577.3 6911.3 4817790.1 17107.0 12983.1 15960.0 2600.7 72 17889.4 13871.1 13692.415151.0 2373.3 120 15298.6 11982.5 11361.5 12880.9 2116.7 168 11752.010982.8 9933.8 10889.5 912.7 240 8921.1 7465.0 6733.4 7706.5 1113.6 3366572.8 5750.5 4687.6 5670.3 945.1 432 1099.6 3425.0 3458.0 2660.9 1352.2528 <80.0 2069.0 2416.2 2242.6 N/A N/A = not applicable

TABLE 60 Plasma concentration (ng/mL) of FP2 measured by LCMS followinga single IV dose in cynomolgus monkeys LC/MS Animal Animal Animal Time(hr) 701 702 703 IV Ave SEM  0 <1000 <1000 <1000 <1000 N/A  1 25340.028820 26220.0 26793.3 1044.7  6 24610.0 26340.0 23810.0 24920.0 746.6 24 18410.0 18680.0 9810.0 15633.3 2912.7  48 16290.0 17370.0 13840.015833.3 1044.3  72 15430.0 14920.0 14280.0 14876.7 332.7 120 11180.011480.0 11440.0 11366.7 94.0 168 9170.0 # # 9170.0 N/A 240 6800.0 7380.07600.0 7260.0 238.6 336 2870.0 3860.0 3480.0 3403.3 288.3 432 — — 3030.03030.0 N/A 528 1150.0 1680.0 1400.0 1410.0 153.1 — = initial run failed,not enough sample for repeat analysis N/A = not applicable # =mislabeled tube, sample excluded from the analysis

TABLE 61 Mean (±SD) pharmacokinetic parameters of FP2 following 1 mg/kgIV and SC administration in cynomolgus monkey. CL or Vz or CL/F Vz/FAUC_(0-last) AUC_(0-inf) t_(1/2) (ml/day/ (ml/ C_(max) T_(max)* (day*(day* Route (day) kg) kg) (ng/ml) (day) ng/ml) ng/ml) SC Mean 8.51 4.656 16178 1.67 180792 221032 SD 1.28 0.8 7 3065 29990 44931 IV Mean 7.054.9 51 28208 0.042 183468 202380 SD 1.45 0.2 12 4082 18268 9617 PKparameters are mean values baser on NCA of immunoassay PK data. *Tmax(median)

Human Plasma Stability Assay

The ex vivo stability of FP2 was examined in fresh heparinized plasma at37° C. for up to 48 hours. Fresh, non-frozen human plasma was generatedfrom heparinized blood from two subjects (one male and one female) bycentrifugation. FP2 was incubated in this matrix at 37° C. wih gentlemixing or 0. 4. 24 and 48 hours. The concentration of FP2 was determineusing an immunoassay method. An independent immunoaffinity capturefollowed by LCMS method was used to quantitate the concentration of theintact dimer present in this matrix under the assay conditions.

In the immunoassay method, the percent recovery from the startingconcentration ranged from 104.8 to 94.1 and did not decrease over time,demonstrating that FP2 is stable in human plasma up to 48 hours ex vivo(FIG. 29 and Table 62). The LCMS method showed that concentrations werestable over time demonstrating that JNJ-64739090 remains an intact dimerin human plasma up to 48 hours ex vivo (FIG. 30 and Table 63).

TABLE 62 Ex vivo stability of FP2 (Normalized Percent Recovery) over 48hours in human plasma (ng/ml) measured by immunoassay. ConcentrationNormalized % Compound Gender Time (hr) (ng/mL) Recovery FP2 female 09104 100.0 4 9056 99.5 24 9332 102.5 48 9374 103.0 male 0 9473 100.0 49929 104.8 24 9081 95.9 48 8912 94.1

TABLE 63 Ex vivo stability of FP2 (Normalized Percent Recovery) over 48hours in human plasma (ng/ml) measured by intact LC/MS. ConcentrationNormalized % Compound Gender Time (hr) (ng/mL) Recovery FP2 female 011090 100.0 4 10830 97.7 24 10500 94.7 48 10030 90.4 male 0 10760 100.04 9640 89.6 24 10190 94.7 48 8500 79.0

Example 19: Efficacy of FP1 and FP2 in Cynomolgus Monkeys

The effects of FP1 and FP2 on food intake and body weight after a singledose in naive cynomolgus monkeys were evaluated.

FP1 was administered subcutaneously to a cohort of naive cynomolgusmonkeys at three dose levels; 1, 3 and 10 nmol/kg. A vehicle treatedgroup was also included. The animals were treated in a blinded manner.The study lasted a total of 6 weeks: 2 weeks of baseline food intakemeasurement and data collection, 4 weeks of data collection after singleadministration of compound. Plasma drug exposures were measured on days1, 7, 14, 21, and 28 following dosing.

Treatment of cynomolgus monkeys with a single dose of FP1 reduced foodintake and body weight compared to vehicle treatment (FIGS. 32-33 ). Asignificant reduction in daily food intake was seen on days 4, 5, 6, and8 through 12 for the 10 nmol/kg dose level (FIG. 32 ). The weeklyaverage of daily food intake was significantly reduced for during week 2post administration for the 10 nmol/kg dose level. The 3 nmol/kg doselevel had a significant percent reduction from the average weekly foodintake prior to dosing on week 2 post administration and the 10 nmol/kgdose level had a significant percent reduction from the average weeklyfood intake prior to dosing in weeks 1 and 2 post administration. Asignificant reduction in percent body weight change from day 0 was seenat day 28 for the 3 nmol/kg dose level, and on day 14, 21, and 28 forthe 10 nmol/kg dose level (FIG. 33 ).

FP2 was administered subcutaneously to a cohort of naïve cynomolgusmonkeys at three dose levels; 1, 3 and 10 nmol/kg. A vehicle treatedgroup was also included. The animals were treated in a blinded manner.The study lasted a total of 11 weeks: 5 weeks of baseline food intakemeasurement and data collection, 1 week of treatment and 5 weeks ofwash-out phase data collection. Plasma drug exposures were measured ondays 1, 7, 14, 21, 28, 35, and 42 following dosing.

Treatment of cynomolgus monkeys with a single dose of FP2 reduced foodintake and body weight compared to vehicle treatment (FIGS. 34-35 ). Asignificant reduction in daily food intake was seen on days 3, 5 through8, 10 and 12 for the 3 nmol/kg dose level and from days 3 through 38 andday 40 for the 10 nmol/kg dose level (FIG. 34 ). The weekly average ofdaily food intake was significantly reduced for week 1 postadministration for the 3 nmol/kg dose level and significantly reducedfor weeks 1 through 6 for the 10 nmol/kg dose level. The 3 nmol/kg doselevel had a significant percent reduction from the week prior to dosingin weekly average daily food intake on week 2 post administration andthe 10 nmol/kg dose level had a significant percent reduction from theweek prior to dosing in weekly average daily food intake on weeks 1through 6 post administration. A significant reduction in percent bodyweight change from day 0 was seen from days 21 through 42 for the 1nmol/kg dose level, from days 14 through 42 for the 3 nmol/kg dose leveland from days 7 through 42 for the 10 nmol/kg dose level (FIG. 34 ).

Example 20: HSA-GDF15:GDF15 Heterodimer

Bioactivity of HSA-GDF15:GDF15 heterodimer was investigated.

To generate an HSA-GDF15:GDF15 heterodimer two constructs were designed.The first construct contained HSA fused to the N-terminus of matureGDF15 (AA 203-308) via a glycine-serine linker (SEQ ID NO: 93). Thesecond construct contained a 6x histidine tagged HSA fused to theN-terminus of mature GDF15 (AA 197-308) via a glycine-serine linker andan HRV3C protease cleavage site (SEQ ID NO: 94). The plasmids wereco-transfected at a 1:1 ratio using the Expi293™ Expression System(Thermo Fisher Scientific) according to the manufacturer's protocol.Peptides were secreted as HSA-GDF15 proteins, including both heterodimerand homodimer forms, wherein monomers were linked by disulfide bonds.

Cell supernatants from transiently transfected Expi293™ cells wereharvested 5 days after transfection, clarified by centrifugation andsterile filtered (0.2 μPES membrane, Corning). Clarified supernatantswere loaded onto a Histrap HP column (GE Healthcare) equilibrated with20 mM sodium phosphate, 500 mM NaCl, pH 7.4. After loading, unboundprotein was removed by washing the column with equilibration buffer.HSA-GDF15 proteins, including both heterodimer and homodimer forms,bound to the column and were eluted with 20 mM sodium phosphate, 150 mMimidazole, pH 7.4. Eluate fractions were pooled and incubated overnightat 4° C. in the presence of 6x histidine tagged HRV3C enzyme (Janssen)to generate the HSA-GDF15:GDF15 heterodimer. Following incubation, theprotein solution was dialyzed into equilibration buffer to removeimidazole before being applied to a HisTrap HP column one more time. TheHSA-GDF15:GDF15 heterodimer eluted in the 20 mM sodium phosphate, 50 mMimidazole, pH 7.4 wash step, while histidine tagged proteins wereretained. The heterodimer was further polished by size exclusionchromatography (SEC) using a HiLoad 26/60 Superdex 200 pg column (GEHealthcare) equilibrated in lx DPBS, pH 7.2. Eluate fractions from theSEC with high purity (determined by SDS-PAGE) of the HSA-GDF15:GDF15heterodimer were pooled and filtered. The protein concentration wasdetermined by absorbance at 280 nm on a BioTek SynergyHTTMspectrophotometer. The quality of the purified protein was assessed bySDS-PAGE and analytical size exclusion HPLC (Ultimate3000 HPLC system).Endotoxin levels were measured using an LAL assay (Pyrotell®-T,Associates of Cape Cod). The purified protein was stored at 4° C.

SK-N-AS cells (ATCC) stably expressing GDF15 receptor (GFRAL) wereseeded in growth medium (10% FBS) in a 96-well plate 24 hours prior tothe assay. After 24 hrs the cells were starved by replacing the culturemedium with 200 μl of DMEM medium supplemented with 1% HI horse serumfor 3 hours in a 37° C. incubator. The 1% HI horse serum supplementedmedium was then replaced with 200 μl of AB1 and incubated for anadditional 2 hours in a 37° C. incubator. To perform the assay, the AB1was aspirated from all wells and 100 μl of variable concentrations ofthe testing compound in AB2 was added and the plate was incubated for 15min in a 37° C. incubator. After 15 minutes, the testing solution wasremoved and 30 μl of lysis buffer (provided in the detection kit) wasadded and the plate was shaken on a plate shaker at room temperature for30 min. For detection, 16 μl of the lysed sample was transferred to a384-well assay plate and 4 μl of HTRF pAKT detection antibodies wereadded. The plate was incubated overnight at room temperature and thenthe HTRF signal read on the Envision (Perkin Elmer).

EC₅₀ values were calculated using GraphPad Prism® Nonlinear Regression(Curve fit). Data are expressed as the Mean± Standard Error (SE) fromthree separate experiments with three replicates per data point.Molecular identity of the HSA-GDF15:GDF15 heterodimer was confirmed bymass spectrometry. The left-shift of the heterodimer curve suggestedthat the HSA-GDF15:GDF15 heterodimer is more potent in inducing pAKTthan the relevant homodimer molecule with an additional albumin.

Example 21: Linker Thermal Stability

Thermal stability was investigated for various linkers that connect HSAand GDF15. To evaluate the potential to fragment and aggregate,HSA-GDF15 fusion proteins with various linkers were diluted to I Omg/ml.After addition of EDTA and Methionine, the samples were incubated under40° C. for 14 days. Then samples were diluted to the concentration of 1mg/ml and evaluated under size-exclusion high-performance liquidchromatography (SE-HPLC). Percent of intact protein as well as aggregateand fragment were quantified for these proteins. Table 64 shows that theHSA-GDF15 proteins with linkers that consist of AP repeats are moststable against fragment under thermal stress.

To evaluate the whether these linker affects GDF15 interaction with itsreceptor, an immunoassay with GFRAL-Fc fusion protein coated on plateand anti-GDF15 or anti-HSA detection was performed, using monoclonalantibodies for GDF15 (Janssen) and HSA (Kerafast, Inc., Boston, Mass.).The assay showed all these linker variants in Table 66 has similarbinding to receptor.

TABLE 64 SE-HPLC results after thermal stress for 14 days. SEQ aggre-frag- ID gate intact ment NO Linker (%) (%) (%) 113 GS(GGGGS)₈ 3.3384.44 12.22 115 GA(GGGGA)₈ 3.51 87.98 8.5 117 (AP)₁₀ 1.64 98.36 0 119(AP)₁₂ 2.36 97.64 0 121 GGS-(EGKSSGSGSESKST)₃-GGS 1.67 85.24 13.09 123GS(PGGGS)₈ 2.96 88.12 8.91 125 GS(AGGGS)₈ 3.44 86.22 10.34 127GGS-(EGKSSGSGSESKST)₂-GGS 1.71 91.17 7.12

While the invention has been described in detail, and with reference tospecific embodiments thereof, it will be apparent to one of ordinaryskill in the art that various changes and modifications can be madetherein without departing from the spirit and scope of the invention.

The sequences referenced in this application are provided in the tablebelow: WT—wild type

WT-wild type SEQ ID NO Description Sequence 1 Human SerumDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNE Albumin, WTVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADC (HSA)CAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL 2 HSA variant,DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV C34STEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL 3 HSA variant,DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNE C34AVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL 5 HSA (C34S)-DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV GS(GGGGS)₄-TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC GDF15 (WT)AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK FusionKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSGGGGSGGGGSGGGGSGGGGSARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI 6 Mature GDF15ARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCI (197-308)GACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQK TDTGVSLQTYDDLLAKDCHCI 7Truncated GDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGAC mature GDF15PSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDT (200-308)GVSLQTYDDLLAKDCHCI 8 TruncatedDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACP mature GDF15SQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTG (201-308)VSLQTYDDLLAKDCHCI 9 TruncatedHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPS mature GDF15QFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGV (202-308)SLQTYDDLLAKDCHCI 10 TruncatedCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQ mature GDF15FRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVS (203-308) LQTYDDLLAKDCHCI11 Truncated CRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMH mature GDF15AQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDL (211-308) LAKDCHCI 25HSA (C34S)- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV AS(GGGGS)₂GT-TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC -GDF15 FusionAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLASGGGGSGGGGSGTARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI 26 HSA (C34S)-DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV AS(GGGGS)₈GT-TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC GDF15 (WT)AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLASGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGTARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVS LQTYDDLLAKDCHCI 27HSA (C34S)- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV AS(AP)₅GT-TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC GDF15 FusionAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLASAPAPAPAPAPGTARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI 28 HSA (C34S)DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV AS(AP)₁₀GT-TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC GDF15 FusionAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLASAPAPAPAPAPAPAPAPAPAPGTARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI 29 HSA (C34S)-DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV AS(AP)₂₀GT-TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC GDF15 FusionAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLASAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPGTARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTY DDLLAKDCHCI 30HSA (C34S)- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV AS(EAAAK)₄GT-TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC GDF15 FusionAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLASEAAAKEAAAKEAAAKEAAAKGTARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI 31 HSA (C34S)-DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV AS(EAAAK)₈GT-TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC GDF15 FusionAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLASEAAAKEAAAKEAAAKEAAAKEAAAKEAAAKEAAAKEAAAKGTARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGV SLQTYDDLLAKDCHCI 36HSA (C34S)- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV GS(GGGGS)₄-TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC GDF15 FusionAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK (deletion 13KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKL mutant)DELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSGGGGSGGGGSGGGGSGGGGSCSRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVL IQKTDTGVSLQTYDDLLAKDCHCI37 HSA (C34S)- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVGS(GGGGS)₄- TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC GDF15 FusionAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK (deletion 14KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKL mutant)DELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSGGGGSGGGGSGGGGSGGGGSCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVL IQKTDTGVSLQTYDDLLAKDCHCI40 HSA (C34S)- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVGS(GGGGS)₄- TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC GDF15 FusionAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSGGGGSGGGGSGGGGSGGGGSARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI 48 HSA (C34A)-DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNE GS(GGGGS)₄-VTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADC GDF15 FusionCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSGGGGSGGGGSGGGGSGGGGSARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI 55 HSA(C34A)-DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNE GS(GGGGS)₈-VTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADC GDF15CAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSL QTYDDLLAKDCHCI 56HSA (C34A)- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNE (AP)₁₀-VTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADC GDF15CAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLAPAPAPAPAPAPAPAPAPAPARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI 59 HSA (C34S)-DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV (AP)₁₀-TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC GDF15AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLAPAPAPAPAPAPAPAPAPAPARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI 60 HSA (C34S)-DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV GS-(GGGGS)₈-TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC GDF15(WT)AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQ TYDDLLAKDCHCI 64HSA (C34S)- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV GS(GGGGS)₄-TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC GDF15 (I89R)AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSGGGGSGGGGSGGGGSGGGGSARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLRQKTDTGVSLQTYDDLLAKDCHCI 65 HSA (C34S)-DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV GS(GGGGS)₄-TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC GDF15 (I89W)AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSGGGGSGGGGSGGGGSGGGGSARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLWQKTDTGVSLQTYDDLLAKDCHCI 66 HSA (C34S)-DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV GS(GGGGS)₄-TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC GDF15 (L34A,AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK S35A, R37A)KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSGGGGSGGGGSGGGGSGGGGSARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVAAPAEVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI 67 HSA (C34S)-DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV GS(GGGGS)₄-TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC GDF15 (V87A,AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK I89A, L98A)KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSGGGGSGGGGSGGGGSGGGGSARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMALAQKTDTGVSAQTYDDLLAKDCHCI 68 HSA (C34S)-DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV GS(GGGGS)₄-TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC GDF15 (L34A,AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK S35A, I89A)KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSGGGGSGGGGSGGGGSGGGGSARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVAAPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLAQKTDTGVSLQTYDDLLAKDCHCI 69 HSA (C34S)-DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV GS(GGGGS)₄-TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC GDF15 (V87A,AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK I89A)KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSGGGGSGGGGSGGGGSGGGGSARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMALAQKTDTGVSLQTYDDLLAKDCHCI 70 HSA (C34S)-DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV GS(GGGGS)₄-TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC GDF15 (Q60W)AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSGGGGSGGGGSGGGGSGGGGSARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAWIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI 71 HSA (C34S)-DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV GS(GGGGS)₄-TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC GDF15 (W32A)AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSGGGGSGGGGSGGGGSGGGGSARNGDHCPLGPGRCCRLHTVRASLEDLGWADAVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI 72 HSA (C34S)-DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV GS(GGGGS)₄-TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC GDF15 (W29A)AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSGGGGSGGGGSGGGGSGGGGSARNGDHCPLGPGRCCRLHTVRASLEDLGAADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI 73 HSA (C34S)-DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV GS(GGGGS)₄-TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC GDF15 (Q60A,AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK S64A, R67A)KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSGGGGSGGGGSGGGGSGGGGSARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAAIKTALHALKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI 74 HSA (C34S)-DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV GS(GGGGS)₄-TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC GDF15 (W29A,AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK Q60A, I61A)KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSGGGGSGGGGSGGGGSGGGGSARNGDHCPLGPGRCCRLHTVRASLEDLGAADWVLSPREVQVTMCIGACPSQFRAANMHAAAKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI 75 HSA (C34S)-DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV GS(GGGGS)₄-TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC GDF15 (W29A,AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK W32A)KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSGGGGSGGGGSGGGGSGGGGSARNGDHCPLGPGRCCRLHTVRASLEDLGAADAVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI 76 nucleic acidgacgcccacaagagcgaggtggcccaccggttcaaggacctgggcgaggagaacttcaaggc encodingcctggtgctgatcgccttcgcccagtacctgcagcagtccccatcgaggaccacgtgaagctggSEQ ID NO: 5tgaacgaggtgaccgagttcgccaagacctgcgtggccgacgagagcgccgagaactgcgacaagagcctgcacaccctgttcggcgacaagctgtgcaccgtggccaccctgcgggagacctacggcgagatggccgactgctgcgccaagcaggagcccgagcggaacgagtgatcctgcagcacaaggacgacaaccccaacctgccccggctggtgcggcccgaggtggacgtgatgtgcaccgccttccacgacaacgaggagaccttcctgaagaagtacctgtacgagatcgcccggcggcacccctacttctacgcccccgagctgctgttcttcgccaagcggtacaaggccgccttcaccgagtgctgccaggccgccgacaaggccgcctgcctgctgcccaagctggacgagctgcgggacgagggcaaggccagcagcgccaagcagcggctgaagtgcgccagcctgcagaagttcggcgagcgggccttcaaggcctgggccgtggcccggctgagccagcggttccccaaggccgagttcgccgaggtgagcaagctggtgaccgacctgaccaaggtgcacaccgagtgctgccacggcgacctgctggagtgcgccgacgaccgggccgacctggccaagtacatctgcgagaaccaggacagcatcagcagcaagctgaaggagtgctgcgagaagcccctgctggagaagagccactgcatcgccgaggtggagaacgacgagatgcccgccgacctgcccagcctggccgccgacttcgtggagagcaaggacgtgtgcaagaactacgccgaggccaaggacgtgttcctgggcatgttcctgtacgagtacgcccggcggcaccccgactacagcgtggtgctgctgctgcggctggccaagacctacgagaccaccctggagaagtgctgcgccgccgccgacccccacgagtgctacgccaaggtgttcgacgagttcaagcccctggtggaggagccccagaacctgatcaagcagaactgcgagctgttcgagcagctgggcgagtacaagttccagaacgccctgctggtgcggtacaccaagaaggtgccccaggtgagcacccccaccctggtggaggtgagccggaacctgggcaaggtgggcagcaagtgctgcaagcaccccgaggccaagcggatgccctgcgccgaggactacctgagcgtggtgctgaaccagctgtgcgtgctgcacgagaagacccccgtgagcgaccgggtgaccaagtgctgcaccgagagcctggtgaaccggcggccctgatcagcgccctggaggtggacgagacctacgtgcccaaggagttcaacgccgagaccttcaccttccacgccgacatctgcaccctgagcgagaaggagcggcagatcaagaagcagaccgccctggtggagctggtgaagcacaagcccaaggccaccaaggagcagctgaaggccgtgatggacgacttcgccgccttcgtggagaagtgctgcaaggccgacgacaaggagacctgcttcgccgaggagggcaagaagctggtggccgccagccaggccgccctgggcctgggcagcggcggcggcggcagcggcggcggcggatctggtggaggtggcagtggaggagggggatccgctcgcaacggtgaccactgccctctgggtcctggtcgctgctgccgcctgcacaccgttcgcgcttctctggaagacctgggttgggctgactgggttctgtctcctcgcgaagttcaggttaccatgtgcatcggtgatgcccttctcagttccgcgctgctaacatgcacgctcagatcaaaacctctctgcaccgcctgaaacctgacaccgttcctgctccttgctgcgttcctgcttcttacaaccctatggttctgatccagaaaaccgacaccggtgtttctctgcagacctacgacgacctgctggctaaagactgccactgc atc 77nucleic acidgatgctcataagtccgaagtcgcccacagattcaaggacctcggagaagaaaattttaaggccctencodingcgtgcttatcgccttcgcccaatacctccagcagtccccgttcgaggaccacgtgaagctcgtgaaSEQ ID NO: 25cgaagtgaccgagtttgccaagacttgtgtggcggatgaatccgccgagaactgcgacaagagcctccacacgctgttcggcgacaagctgtgcaccgtcgccacgctgagagaaacttacggagagatggccgactgctgcgcaaagcaggagccggaacggaacgaatgcttcctgcaacataaggacgataaccctaacttgcctcgcctggtccgccctgaggtcgacgtgatgtgcaccgcgttccacgacaacgaggaaacctttcttaagaagtacctgtacgagattgcgcggaggcacccttatttctacgcccccgaactgttgttcttcgccaagcggtacaaggctgcctttaccgaatgctgccaggccgccgataaggcggcttgcctgctgccgaagctcgacgagttgcgcgatgaggggaaggcgtcctccgctaagcagcggctgaaatgtgcgagcctccagaagttcggggagcgcgccttcaaggcctgggccgtggcgcgcctgtctcaacggttcccgaaggccgagttcgccgaagtgtcgaagctggtcaccgacctgacgaaagtgcacaccgaatgttgtcacggcgatctgctggaatgcgccgatgacagagccgatttggccaagtacatctgcgaaaaccaggacagcatttcgtcaaagctgaaggaatgctgcgaaaagcccttgctggaaaagtcccactgcatcgcggaagtggagaacgacgagatgcccgccgacctcccgtccctggccgccgatttcgtggagtcgaaggatgtgtgcaagaactacgcagaagccaaggacgtgttcctgggaatgtttctgtatgagtacgcccgccgccacccggactactcggtcgtgctcctgctgcgactggcaaagacctacgaaaccactctggagaagtgctgcgccgccgcggacccgcacgagtgctacgcaaaggtgttcgacgagttcaagccacttgtcgaggagcctcagaacctgatcaagcagaactgcgaactgttcgagcagctgggagagtacaaattccagaacgcgcttctcgtgcgctacaccaagaaggtcccccaggtgtccactccgaccctggtggaagtgtccaggaacctgggaaaggtcggctccaagtgttgcaagcatcccgaggctaagcgcatgccctgcgccgaggactacttgtccgtggtgctgaatcagctgtgcgtgctccatgaaaagaccccagtgtccgacagagtgaccaagtgctgtaccgaatcgctcgtgaaccggcggccgtgcttttccgcactggaggtggacgaaacctacgtgccgaaggagttcaacgcagaaaccttcactttccacgccgacatctgcactctgtccgagaaggagcggcagattaagaagcagactgccctggtggagcttgtgaaacacaagcctaaggccaccaaagagcagctgaaggccgtcatggatgatttcgcggccttcgtggaaaagtgttgtaaagcggacgacaaggagacttgcttcgccgaagaaggaaagaagctcgtggcagcgtcacaggccgctctgggcctcgctagcggtggagggggcagcggtggtggaggatccggtaccgcgcgcaacggggaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata 78 nucleic acidgatgctcataagtccgaagtcgcccacagattcaaggacctcggagaagaaaattttaaggccctencodingcgtgcttatcgccttcgcccaatacctccagcagtccccgttcgaggaccacgtgaagctcgtgaaSEQ ID NO: 26cgaagtgaccgagtttgccaagacttgtgtggcggatgaatccgccgagaactgcgacaagagcctccacacgctgttcggcgacaagctgtgcaccgtcgccacgctgagagaaacttacggagagatggccgactgctgcgcaaagcaggagccggaacggaacgaatgcttcctgcaacataaggacgataaccctaacttgcctcgcctggtccgccctgaggtcgacgtgatgtgcaccgcgttccacgacaacgaggaaacctttcttaagaagtacctgtacgagattgcgcggaggcacccttatttctacgcccccgaactgttgttcttcgccaagcggtacaaggctgcctttaccgaatgctgccaggccgccgataaggcggcttgcctgctgccgaagctcgacgagttgcgcgatgaggggaaggcgtcctccgctaagcagcggctgaaatgtgcgagcctccagaagttcggggagcgcgccttcaaggcctgggccgtggcgcgcctgtctcaacggttcccgaaggccgagttcgccgaagtgtcgaagctggtcaccgacctgacgaaagtgcacaccgaatgttgtcacggcgatctgctggaatgcgccgatgacagagccgatttggccaagtacatctgcgaaaaccaggacagcatttcgtcaaagctgaaggaatgctgcgaaaagcccttgctggaaaagtcccactgcatcgcggaagtggagaacgacgagatgcccgccgacctcccgtccctggccgccgatttcgtggagtcgaaggatgtgtgcaagaactacgcagaagccaaggacgtgttcctgggaatgtttctgtatgagtacgcccgccgccacccggactactcggtcgtgctcctgctgcgactggcaaagacctacgaaaccactctggagaagtgctgcgccgccgcggacccgcacgagtgctacgcaaaggtgttcgacgagttcaagccacttgtcgaggagcctcagaacctgatcaagcagaactgcgaactgttcgagcagctgggagagtacaaattccagaacgcgcttctcgtgcgctacaccaagaaggtcccccaggtgtccactccgaccctggtggaagtgtccaggaacctgggaaaggtcggctccaagtgttgcaagcatcccgaggctaagcgcatgccctgcgccgaggactacttgtccgtggtgctgaatcagctgtgcgtgctccatgaaaagaccccagtgtccgacagagtgaccaagtgctgtaccgaatcgctcgtgaaccggcggccgtgcttttccgcactggaggtggacgaaacctacgtgccgaaggagttcaacgcagaaaccttcactttccacgccgacatctgcactctgtccgagaaggagcggcagattaagaagcagactgccctggtggagcttgtgaaacacaagcctaaggccaccaaagagcagctgaaggccgtcatggatgatttcgcggccttcgtggaaaagtgttgtaaagcggacgacaaggagacttgatcgccgaagaaggaaagaagctcgtggcagcgtcacaggccgctctgggcctcgctagcggaggtggcggatcaggtggcggaggtagcggtggaggcggctctggcggaggtggatcaggcggaggaggttccggtggaggaggctcaggaggaggaggaagtggaggagggggatccggtaccgcgcgcaacggggaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata 79 nucleic acidgatgctcataagtccgaagtcgcccacagattcaaggacctcggagaagaaaattttaaggccctencodingcgtgcttatcgccttcgcccaatacctccagcagtccccgttcgaggaccacgtgaagctcgtgaaSEQ ID NO: 27cgaagtgaccgagtttgccaagacttgtgtggcggatgaatccgccgagaactgcgacaagagcctccacacgctgttcggcgacaagctgtgcaccgtcgccacgctgagagaaacttacggagagatggccgactgctgcgcaaagcaggagccggaacggaacgaatgcttcctgcaacataaggacgataaccctaacttgcctcgcctggtccgccctgaggtcgacgtgatgtgcaccgcgttccacgacaacgaggaaacctttcttaagaagtacctgtacgagattgcgcggaggcacccttatttctacgcccccgaactgttgttcttcgccaagcggtacaaggctgcctttaccgaatgctgccaggccgccgataaggcggcttgcctgctgccgaagctcgacgagttgcgcgatgaggggaaggcgtcctccgctaagcagcggctgaaatgtgcgagcctccagaagttcggggagcgcgccttcaaggcctgggccgtggcgcgcctgtctcaacggttcccgaaggccgagttcgccgaagtgtcgaagctggtcaccgacctgacgaaagtgcacaccgaatgttgtcacggcgatctgctggaatgcgccgatgacagagccgatttggccaagtacatctgcgaaaaccaggacagcatttcgtcaaagctgaaggaatgctgcgaaaagcccttgctggaaaagtcccactgcatcgcggaagtggagaacgacgagatgcccgccgacctcccgtccctggccgccgatttcgtggagtcgaaggatgtgtgcaagaactacgcagaagccaaggacgtgttcctgggaatgtttctgtatgagtacgcccgccgccacccggactactcggtcgtgctcctgctgcgactggcaaagacctacgaaaccactctggagaagtgctgcgccgccgcggacccgcacgagtgctacgcaaaggtgttcgacgagttcaagccacttgtcgaggagcctcagaacctgatcaagcagaactgcgaactgttcgagcagctgggagagtacaaattccagaacgcgcttctcgtgcgctacaccaagaaggtcccccaggtgtccactccgaccctggtggaagtgtccaggaacctgggaaaggtcggctccaagtgttgcaagcatcccgaggctaagcgcatgccctgcgccgaggactacttgtccgtggtgctgaatcagctgtgcgtgctccatgaaaagaccccagtgtccgacagagtgaccaagtgctgtaccgaatcgctcgtgaaccggcggccgtgcttttccgcactggaggtggacgaaacctacgtgccgaaggagttcaacgcagaaaccttcactttccacgccgacatctgcactctgtccgagaaggagcggcagattaagaagcagactgccctggtggagcttgtgaaacacaagcctaaggccaccaaagagcagctgaaggccgtcatggatgatttcgcggccttcgtggaaaagtgttgtaaagcggacgacaaggagacttgatcgccgaagaaggaaagaagctcgtggcagcgtcacaggccgctctgggcctcgctagcgcacctgcccccgctccagctcctgcaccaggtaccgcgcgcaacggggaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata 80 nucleic acidgatgctcataagtccgaagtcgcccacagattcaaggacctcggagaagaaaattttaaggccctencodingcgtgcttatcgccttcgcccaatacctccagcagtccccgttcgaggaccacgtgaagctcgtgaaSEQ ID NO: 28cgaagtgaccgagtttgccaagacttgtgtggcggatgaatccgccgagaactgcgacaagagcctccacacgctgttcggcgacaagctgtgcaccgtcgccacgctgagagaaacttacggagagatggccgactgctgcgcaaagcaggagccggaacggaacgaatgcttcctgcaacataaggacgataaccctaacttgcctcgcctggtccgccctgaggtcgacgtgatgtgcaccgcgttccacgacaacgaggaaacctttcttaagaagtacctgtacgagattgcgcggaggcacccttatttctacgcccccgaactgttgttcttcgccaagcggtacaaggctgcctttaccgaatgctgccaggccgccgataaggcggcttgcctgctgccgaagctcgacgagttgcgcgatgaggggaaggcgtcctccgctaagcagcggctgaaatgtgcgagcctccagaagttcggggagcgcgccttcaaggcctgggccgtggcgcgcctgtctcaacggttcccgaaggccgagttcgccgaagtgtcgaagctggtcaccgacctgacgaaagtgcacaccgaatgttgtcacggcgatctgctggaatgcgccgatgacagagccgatttggccaagtacatctgcgaaaaccaggacagcatttcgtcaaagctgaaggaatgctgcgaaaagcccttgctggaaaagtcccactgcatcgcggaagtggagaacgacgagatgcccgccgacctcccgtccctggccgccgatttcgtggagtcgaaggatgtgtgcaagaactacgcagaagccaaggacgtgttcctgggaatgtttctgtatgagtacgcccgccgccacccggactactcggtcgtgctcctgctgcgactggcaaagacctacgaaaccactctggagaagtgctgcgccgccgcggacccgcacgagtgctacgcaaaggtgttcgacgagttcaagccacttgtcgaggagcctcagaacctgatcaagcagaactgcgaactgttcgagcagctgggagagtacaaattccagaacgcgcttctcgtgcgctacaccaagaaggtcccccaggtgtccactccgaccctggtggaagtgtccaggaacctgggaaaggtcggctccaagtgttgcaagcatcccgaggctaagcgcatgccctgcgccgaggactacttgtccgtggtgctgaatcagctgtgcgtgctccatgaaaagaccccagtgtccgacagagtgaccaagtgctgtaccgaatcgctcgtgaaccggcggccgtgcttttccgcactggaggtggacgaaacctacgtgccgaaggagttcaacgcagaaaccttcactttccacgccgacatctgcactctgtccgagaaggagcggcagattaagaagcagactgccctggtggagcttgtgaaacacaagcctaaggccaccaaagagcagctgaaggccgtcatggatgatttcgcggccttcgtggaaaagtgttgtaaagcggacgacaaggagacttgatcgccgaagaaggaaagaagctcgtggcagcgtcacaggccgctctgggcctcgctagcgcacctgcccccgctccagcacccgccccagcccctgctcccgcaccagctcctgcaccaggtaccgctcgcaacggtgaccactgccctctgggtcctggtcgctgctgccgcctgcacaccgttcgcgcttctctggaagacctgggttgggctgactgggttctgtctcctcgcgaagttcaggttaccatgtgcatcggtgcttgcccttctcagttccgcgctgctaacatgcacgctcagatcaaaacctctctgcaccgcctgaaacctgacaccgttcctgctccttgctgcgttcctgcttcttacaaccctatggttctgatccagaaaaccgacaccggtgtttctctgcagacctacgacgacctgctggctaaagactgccactgcatc 81 nucleic acidgatgctcataagtccgaagtcgcccacagattcaaggacctcggagaagaaaattttaaggccctencodingcgtgcttatcgccttcgcccaatacctccagcagtccccgttcgaggaccacgtgaagctcgtgaaSEQ ID NO: 29cgaagtgaccgagtttgccaagacttgtgtggcggatgaatccgccgagaactgcgacaagagcctccacacgctgttcggcgacaagctgtgcaccgtcgccacgctgagagaaacttacggagagatggccgactgctgcgcaaagcaggagccggaacggaacgaatgcttcctgcaacataaggacgataaccctaacttgcctcgcctggtccgccctgaggtcgacgtgatgtgcaccgcgttccacgacaacgaggaaacctttcttaagaagtacctgtacgagattgcgcggaggcacccttatttctacgcccccgaactgttgttcttcgccaagcggtacaaggctgcctttaccgaatgctgccaggccgccgataaggcggcttgcctgctgccgaagctcgacgagttgcgcgatgaggggaaggcgtcctccgctaagcagcggctgaaatgtgcgagcctccagaagttcggggagcgcgccttcaaggcctgggccgtggcgcgcctgtctcaacggttcccgaaggccgagttcgccgaagtgtcgaagctggtcaccgacctgacgaaagtgcacaccgaatgttgtcacggcgatctgctggaatgcgccgatgacagagccgatttggccaagtacatctgcgaaaaccaggacagcatttcgtcaaagctgaaggaatgctgcgaaaagcccttgctggaaaagtcccactgcatcgcggaagtggagaacgacgagatgcccgccgacctcccgtccctggccgccgatttcgtggagtcgaaggatgtgtgcaagaactacgcagaagccaaggacgtgttcctgggaatgtttctgtatgagtacgcccgccgccacccggactactcggtcgtgctcctgctgcgactggcaaagacctacgaaaccactctggagaagtgctgcgccgccgcggacccgcacgagtgctacgcaaaggtgttcgacgagttcaagccacttgtcgaggagcctcagaacctgatcaagcagaactgcgaactgttcgagcagctgggagagtacaaattccagaacgcgcttctcgtgcgctacaccaagaaggtcccccaggtgtccactccgaccctggtggaagtgtccaggaacctgggaaaggtcggctccaagtgttgcaagcatcccgaggctaagcgcatgccctgcgccgaggactacttgtccgtggtgctgaatcagctgtgcgtgctccatgaaaagaccccagtgtccgacagagtgaccaagtgctgtaccgaatcgctcgtgaaccggcggccgtgcttttccgcactggaggtggacgaaacctacgtgccgaaggagttcaacgcagaaaccttcactttccacgccgacatctgcactctgtccgagaaggagcggcagattaagaagcagactgccctggtggagcttgtgaaacacaagcctaaggccaccaaagagcagctgaaggccgtcatggatgatttcgcggccttcgtggaaaagtgttgtaaagcggacgacaaggagacttgatcgccgaagaaggaaagaagctcgtggcagcgtcacaggccgctctgggcctcgctagcgcacctgcccccgctccagccccagctcctgcacctgctccagcaccagctcctgcaccagctccagcccctgcacctgcacccgctccagccccagctcctgcacctgctccagcaccaggtaccgcgcgcaacggggaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata 82 nucleic acidgatgctcataagtccgaagtcgcccacagattcaaggacctcggagaagaaaattttaaggccctencodingcgtgcttatcgccttcgcccaatacctccagcagtccccgttcgaggaccacgtgaagctcgtgaaSEQ ID NO: 30cgaagtgaccgagtttgccaagacttgtgtggcggatgaatccgccgagaactgcgacaagagcctccacacgctgttcggcgacaagctgtgcaccgtcgccacgctgagagaaacttacggagagatggccgactgctgcgcaaagcaggagccggaacggaacgaatgcttcctgcaacataaggacgataaccctaacttgcctcgcctggtccgccctgaggtcgacgtgatgtgcaccgcgttccacgacaacgaggaaacctttcttaagaagtacctgtacgagattgcgcggaggcacccttatttctacgcccccgaactgttgttcttcgccaagcggtacaaggctgcctttaccgaatgctgccaggccgccgataaggcggcttgcctgctgccgaagctcgacgagttgcgcgatgaggggaaggcgtcctccgctaagcagcggctgaaatgtgcgagcctccagaagttcggggagcgcgccttcaaggcctgggccgtggcgcgcctgtctcaacggttcccgaaggccgagttcgccgaagtgtcgaagctggtcaccgacctgacgaaagtgcacaccgaatgttgtcacggcgatctgctggaatgcgccgatgacagagccgatttggccaagtacatctgcgaaaaccaggacagcatttcgtcaaagctgaaggaatgctgcgaaaagcccttgctggaaaagtcccactgcatcgcggaagtggagaacgacgagatgcccgccgacctcccgtccctggccgccgatttcgtggagtcgaaggatgtgtgcaagaactacgcagaagccaaggacgtgttcctgggaatgtttctgtatgagtacgcccgccgccacccggactactcggtcgtgctcctgctgcgactggcaaagacctacgaaaccactctggagaagtgctgcgccgccgcggacccgcacgagtgctacgcaaaggtgttcgacgagttcaagccacttgtcgaggagcctcagaacctgatcaagcagaactgcgaactgttcgagcagctgggagagtacaaattccagaacgcgcttctcgtgcgctacaccaagaaggtcccccaggtgtccactccgaccctggtggaagtgtccaggaacctgggaaaggtcggctccaagtgttgcaagcatcccgaggctaagcgcatgccctgcgccgaggactacttgtccgtggtgctgaatcagctgtgcgtgctccatgaaaagaccccagtgtccgacagagtgaccaagtgctgtaccgaatcgctcgtgaaccggcggccgtgcttttccgcactggaggtggacgaaacctacgtgccgaaggagttcaacgcagaaaccttcactttccacgccgacatctgcactctgtccgagaaggagcggcagattaagaagcagactgccctggtggagcttgtgaaacacaagcctaaggccaccaaagagcagctgaaggccgtcatggatgatttcgcggccttcgtggaaaagtgttgtaaagcggacgacaaggagacttgatcgccgaagaaggaaagaagctcgtggcagcgtcacaggccgctctgggcctcgctagcgaagcagcagccaaagaagcagccgcaaaagaagcagccgctaaggaggccgcagcaaagggtaccgcgcgcaacggggaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata 83 nucleic acidgacgcccacaagagcgaggtggcccaccggttcaaggacctgggcgaggagaacttcaaggc encodingcctggtgctgatcgccttcgcccagtacctgcagcagtccccatcgaggaccacgtgaagctggSEQ ID NO: 40tgaacgaggtgaccgagttcgccaagacctgcgtggccgacgagagcgccgagaactgcgacaagagcctgcacaccctgttcggcgacaagctgtgcaccgtggccaccctgcgggagacctacggcgagatggccgactgctgcgccaagcaggagcccgagcggaacgagtgatcctgcagcacaaggacgacaaccccaacctgccccggctggtgcggcccgaggtggacgtgatgtgcaccgccttccacgacaacgaggagaccttcctgaagaagtacctgtacgagatcgcccggcggcacccctacttctacgcccccgagctgctgttcttcgccaagcggtacaaggccgccttcaccgagtgctgccaggccgccgacaaggccgcctgcctgctgcccaagctggacgagctgcgggacgagggcaaggccagcagcgccaagcagcggctgaagtgcgccagcctgcagaagttcggcgagcgggccttcaaggcctgggccgtggcccggctgagccagcggttccccaaggccgagttcgccgaggtgagcaagctggtgaccgacctgaccaaggtgcacaccgagtgctgccacggcgacctgctggagtgcgccgacgaccgggccgacctggccaagtacatctgcgagaaccaggacagcatcagcagcaagctgaaggagtgctgcgagaagcccctgctggagaagagccactgcatcgccgaggtggagaacgacgagatgcccgccgacctgcccagcctggccgccgacttcgtggagagcaaggacgtgtgcaagaactacgccgaggccaaggacgtgttcctgggcatgttcctgtacgagtacgcccggcggcaccccgactacagcgtggtgctgctgctgcggctggccaagacctacgagaccaccctggagaagtgctgcgccgccgccgacccccacgagtgctacgccaaggtgttcgacgagttcaagcccctggtggaggagccccagaacctgatcaagcagaactgcgagctgttcgagcagctgggcgagtacaagttccagaacgccctgctggtgcggtacaccaagaaggtgccccaggtgagcacccccaccctggtggaggtgagccggaacctgggcaaggtgggcagcaagtgctgcaagcaccccgaggccaagcggatgccctgcgccgaggactacctgagcgtggtgctgaaccagctgtgcgtgctgcacgagaagacccccgtgagcgaccgggtgaccaagtgctgcaccgagagcctggtgaaccggcggccctgatcagcgccctggaggtggacgagacctacgtgcccaaggagttcaacgccgagaccttcaccttccacgccgacatctgcaccctgagcgagaaggagcggcagatcaagaagcagaccgccctggtggagctggtgaagcacaagcccaaggccaccaaggagcagctgaaggccgtgatggacgacttcgccgccttcgtggagaagtgctgcaaggccgacgacaaggagacctgcttcgccgaggagggcaagaagctggtggccgccagccaggccgccctgggcctgggcagcggcggcggcggcagcggcggcggcggatctggtggaggtggcagtggaggagggggatccgcgcgcaacggggaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata 84 nucleic acidgatgcacacaagagtgaggttgctcatcggtttaaagatttgggagaagaaaatttcaaagccttgencodinggtgttgattgcctttgctcagtatcttcagcaggccccatttgaagatcatgtaaaattagtgaatgaaSEQ ID NO: 55gtaactgaatttgcaaaaacatgtgttgctgatgagtcagctgaaaattgtgacaaatcacttcataccctttttggagacaaattatgcacagttgcaactcttcgtgaaacctatggtgaaatggctgactgctgtgcaaaacaagaacctgagagaaatgaatgcttcttgcaacacaaagatgacaacccaaacctcccccgattggtgagaccagaggttgatgtgatgtgcactgcttttcatgacaatgaagagacatttttgaaaaaatacttatatgaaattgccagaagacatccttacttttatgccccggaactccttttctttgctaaaaggtataaagctgcttttacagaatgttgccaagctgctgataaagctgcctgcctgttgccaaagctcgatgaacttcgggatgaagggaaggcttcgtctgccaaacagagactcaagtgtgccagtctccaaaaatttggagaaagagctttcaaagcatgggcagtagctcgcctgagccagagatttcccaaagctgagtttgcagaagtttccaagttagtgacagatcttaccaaagtccacacggaatgctgccatggagatctgcttgaatgtgctgatgacagggcggaccttgccaagtatatctgtgaaaatcaagattcgatctccagtaaactgaaggaatgctgtgaaaaacctctgttggaaaaatcccactgcattgccgaagtggaaaatgatgagatgcctgctgacttgccttcattagctgctgattttgttgaaagtaaggatgtttgcaaaaactatgctgaggcaaaggatgtcttcctgggcatgtttttgtatgaatatgcaagaaggcatcctgattactctgtcgtgctgctgctgagacttgccaagacatatgaaaccactctagagaagtgctgtgccgctgcagatcctcatgaatgctatgccaaagtgttcgatgaatttaaacctatgtggaagagcctcagaatttaatcaaacaaaattgtgagctttttgagcagcttggagagtacaaattccagaatgcgctattagttcgttacaccaagaaagtaccccaagtgtcaactccaactcttgtagaggtctcaagaaacctaggaaaagtgggcagcaaatgttgtaaacatcctgaagcaaaaagaatgccctgtgcagaagactatctatccgtggtcctgaaccagttatgtgtgttgcatgagaaaacgccagtaagtgacagagtcaccaaatgctgcacagaatccttggtgaacaggcgaccatgcttttcagctctggaagtcgatgaaacatacgttcccaaagagtttaatgctgaaacattcaccttccatgcagatatatgcacactttctgagaaggagagacaaatcaagaaacaaactgcacttgttgagctcgtgaaacacaagcccaaggcaacaaaagagcaactgaaagctgttatggatgatttcgcagatttgtagagaagtgctgcaaggctgacgataaggagacctgattgccgaggagggtaaaaaacttgttgctgcaagtcaagctgccttaggcttaggcagcggcggcggcggcagcggcggcggcggatctggtggaggtggcagtggaggagggggatccggcggcggcggcagcggcggcggcggatctggtggaggtggcagtggaggagggggatccgcgcgcaacggggaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata 85 nucleic acidgatgcacacaagagtgaggttgctcatcggtttaaagatttgggagaagaaaatttcaaagccttgencodinggtgttgattgcctttgctcagtatcttcagcaggccccatttgaagatcatgtaaaattagtgaatgaaSEQ ID NO: 56gtaactgaatttgcaaaaacatgtgttgctgatgagtcagctgaaaattgtgacaaatcacttcataccctttttggagacaaattatgcacagttgcaactcttcgtgaaacctatggtgaaatggctgactgctgtgcaaaacaagaacctgagagaaatgaatgcttcttgcaacacaaagatgacaacccaaacctcccccgattggtgagaccagaggttgatgtgatgtgcactgcttttcatgacaatgaagagacatttttgaaaaaatacttatatgaaattgccagaagacatccttacttttatgccccggaactccttttctttgctaaaaggtataaagctgcttttacagaatgttgccaagctgctgataaagctgcctgcctgttgccaaagctcgatgaacttcgggatgaagggaaggcttcgtctgccaaacagagactcaagtgtgccagtctccaaaaatttggagaaagagctttcaaagcatgggcagtagctcgcctgagccagagatttcccaaagctgagtttgcagaagtttccaagttagtgacagatcttaccaaagtccacacggaatgctgccatggagatctgcttgaatgtgctgatgacagggcggaccttgccaagtatatctgtgaaaatcaagattcgatctccagtaaactgaaggaatgctgtgaaaaacctctgttggaaaaatcccactgcattgccgaagtggaaaatgatgagatgcctgctgacttgccttcattagctgctgattttgttgaaagtaaggatgtttgcaaaaactatgctgaggcaaaggatgtcttcctgggcatgtttttgtatgaatatgcaagaaggcatcctgattactctgtcgtgctgctgctgagacttgccaagacatatgaaaccactctagagaagtgctgtgccgctgcagatcctcatgaatgctatgccaaagtgttcgatgaatttaaacctcttgtggaagagcctcagaatttaatcaaacaaaattgtgagctttttgagcagcttggagagtacaaattccagaatgcgctattagttcgttacaccaagaaagtaccccaagtgtcaactccaactcttgtagaggtctcaagaaacctaggaaaagtgggcagcaaatgttgtaaacatcctgaagcaaaaagaatgccctgtgcagaagactatctatccgtggtcctgaaccagttatgtgtgttgcatgagaaaacgccagtaagtgacagagtcaccaaatgctgcacagaatccttggtgaacaggcgaccatgcttttcagctctggaagtcgatgaaacatacgttcccaaagagtttaatgctgaaacattcaccttccatgcagatatatgcacactttctgagaaggagagacaaatcaagaaacaaactgcacttgttgagctcgtgaaacacaagcccaaggcaacaaaagagcaactgaaagctgttatggatgatttcgcagcttttgtagagaagtgctgcaaggctgacgataaggagacctgattgccgaggagggtaaaaaacttgttgctgcaagtcaagctgccttaggcttagcacctgcccccgctccagcacccgccccagcccctgctcccgcaccagctcctgcaccagcgcgcaacggggaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata 86 nucleic acidgatgcacacaagagtgaggttgctcatcggtttaaagatttgggagaagaaaatttcaaagccttgencodinggtgttgattgcctttgctcagtatcttcagcagtccccatttgaagatcatgtaaaattagtgaatgaagSEQ ID NO: 40taactgaatttgcaaaaacatgtgttgctgatgagtcagctgaaaattgtgacaaatcacttcatacc(Codonctttttggagacaaattatgcacagttgcaactcttcgtgaaacctatggtgaaatggctgactgctgtoptimization 1)gcaaaacaagaacctgagagaaatgaatgcttcttgcaacacaaagatgacaacccaaacctcccccgattggtgagaccagaggttgatgtgatgtgcactgcttttcatgacaatgaagagacatttttgaaaaaatacttatatgaaattgccagaagacatccttacttttatgccccggaactccttttctttgctaaaaggtataaagctgcttttacagaatgttgccaagctgctgataaagctgcctgcctgttgccaaagctcgatgaacttcgggatgaagggaaggcttcgtctgccaaacagagactcaagtgtgccagtctccaaaaatttggagaaagagctttcaaagcatgggcagtagctcgcctgagccagagatttcccaaagctgagtttgcagaagtttccaagttagtgacagatcttaccaaagtccacacggaatgctgccatggagatctgcttgaatgtgctgatgacagggcggaccttgccaagtatatctgtgaaaatcaagattcgatctccagtaaactgaaggaatgctgtgaaaaacctctgttggaaaaatcccactgcattgccgaagtggaaaatgatgagatgcctgctgacttgccttcattagctgctgattttgttgaaagtaaggatgtttgcaaaaactatgctgaggcaaaggatgtcttcctgggcatgtttttgtatgaatatgcaagaaggcatcctgattactctgtcgtgctgctgctgagacttgccaagacatatgaaaccactctagagaagtgctgtgccgctgcagatcctcatgaatgctatgccaaagtgttcgatgaatttaaacctcttgtggaagagcctcagaatttaatcaaacaaaattgtgagattttgagcagatggagagtacaaattccagaatgcgctattagttcgttacaccaagaaagtaccccaagtgtcaactccaactatgtagaggtctcaagaaacctaggaaaagtgggcagcaaatgttgtaaacatcctgaagcaaaaagaatgccctgtgcagaagactatctatccgtggtcctgaaccagttatgtgtgttgcatgagaaaacgccagtaagtgacagagtcaccaaatgctgcacagaatccttggtgaacaggcgaccatgcttttcagctctggaagtcgatgaaacatacgttcccaaagagtttaatgctgaaacattcaccttccatgcagatatatgcacactttctgagaaggagagacaaatcaagaaacaaactgcacttgttgagctcgtgaaacacaagcccaaggcaacaaaagagcaactgaaagctgttatggatgatttcgcagcttttgtagagaagtgctgcaaggctgacgataaggagacctgctttgccgaggagggtaaaaaacttgttgctgcaagtcaagctgccttaggcttaggcagcggcggcggcggcagcggcggcggcggatctggtggaggtggcagtggaggagggggatccgcgcgcaacggggaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata 87 nucleic acidgacgcccacaagagcgaggtggcccacagattcaaggacctgggcgaggaaaacttcaaggc encodingcctggtgctgatcgccttcgcccagtacctgcagcagagcccatcgaggaccacgtgaagctggSEQ ID NO: 40tcaacgaagtgaccgagttcgccaagacctgcgtggccgacgagagcgccgagaactgcgaca (Codonagagcctgcacaccctgttcggcgacaagctgtgcaccgtggccaccctgcgggaaacctacgoptimization 2)gcgagatggccgactgctgcgccaagcaggaacccgagcggaacgagtgcttcctgcagcacaaggacgacaaccccaacctgcccagactcgtgcggcccgaggtggacgtgatgtgcaccgccttccacgacaacgaggaaaccttcctgaagaagtacctgtacgagatcgccagacggcacccctacttctacgcccccgagctgctgttcttcgccaagcggtacaaggccgccttcaccgagtgctgccaggccgccgataaggccgcctgcctgctgcccaagctggacgagctgagagatgagggcaaggccagctccgccaagcagcggctgaagtgcgccagcctgcagaagttcggcgagcgggcctttaaggcttgggctgtggcccggctgagccagagattccccaaggccgagtttgccgaggtgtccaagctggtcaccgacctgaccaaggtgcacaccgagtgttgtcacggcgacctgctggaatgcgccgacgacagagccgacctggccaagtacatctgcgagaaccaggacagcatcagcagcaagctgaaagagtgctgcgagaagcccctgctggaaaagagccactgtatcgccgaggtggaaaacgacgagatgcccgctgacctgcccagcctggccgccgacttcgtggaaagcaaggacgtgtgcaagaactacgccgaggccaaggatgtgttcctgggcatgttcctgtatgagtacgcccgcagacaccccgactacagcgtggtgctgctgctgcggctggccaagacctacgagacaaccctggaaaagtgctgcgccgctgccgacccccacgagtgctacgccaaggtgttcgacgagttcaagcctctggtggaagaaccccagaacctgatcaagcagaactgcgagctgttcgagcagctgggcgagtacaagttccagaacgccctgctcgtgcggtacaccaagaaagtgccccaggtgtccacccccaccctggtcgaagtgtcccggaacctgggcaaagtgggcagcaagtgctgcaagcaccctgaggccaagcggatgccctgcgccgaggactacctgtccgtggtgctgaaccagctgtgcgtgctgcacgagaaaacccccgtgtccgacagagtgaccaagtgctgtaccgagagcctggtcaacagacggccctgcttcagcgccctggaagtggacgagacatacgtgcccaaagagttcaacgccgagacattcaccttccacgccgacatctgcaccctgagcgagaaagagcggcagatcaagaagcagaccgccctggtcgagctggtcaagcacaagcccaaggccaccaaagaacagctgaaggccgtgatggacgacttcgccgccttcgtcgagaagtgttgcaaggccgacgacaaagagacatgcttcgccgaagagggcaagaaactggtggccgcctctcaggccgccctgggactgggatctggcggcggaggaagcggaggcggaggatctgggggaggcggctctggcggagggggatccgccagaaatggcgaccactgtcccctgggccctggccggtgttgcagactgcacacagtgcgggccagcctggaagatctgggctgggccgattgggtgctgagccccagagaagtgcaggtcacaatgtgcatcggcgcctgccccagccagttcagagccgccaacatgcacgcccagatcaagaccagcctgcaccggctgaagcccgacaccgtgcctgccccttgttgcgtgcccgccagctacaaccccatggtgctgattcagaaaaccgacaccggcgtgtccctgcagacctacgacgatctgctggccaaggactgccactgcatc 88nucleic acidgatgcacacaagagtgaggttgctcatcggtttaaagatttgggagaagaaaatttcaaagccttgencodinggtgttgattgcctttgctcagtatcttcagcaggccccatttgaagatcatgtaaaattagtgaatgaaSEQ ID NO: 48gtaactgaatttgcaaaaacatgtgttgctgatgagtcagctgaaaattgtgacaaatcacttcataccctttttggagacaaattatgcacagttgcaactcttcgtgaaacctatggtgaaatggctgactgctgtgcaaaacaagaacctgagagaaatgaatgcttcttgcaacacaaagatgacaacccaaacctcccccgattggtgagaccagaggttgatgtgatgtgcactgcttttcatgacaatgaagagacatttttgaaaaaatacttatatgaaattgccagaagacatccttacttttatgccccggaactccttttctttgctaaaaggtataaagctgcttttacagaatgttgccaagctgctgataaagctgcctgcctgttgccaaagctcgatgaacttcgggatgaagggaaggcttcgtctgccaaacagagactcaagtgtgccagtctccaaaaatttggagaaagagctttcaaagcatgggcagtagctcgcctgagccagagatttcccaaagctgagtttgcagaagtttccaagttagtgacagatcttaccaaagtccacacggaatgctgccatggagatctgcttgaatgtgctgatgacagggcggaccttgccaagtatatctgtgaaaatcaagattcgatctccagtaaactgaaggaatgctgtgaaaaacctctgttggaaaaatcccactgcattgccgaagtggaaaatgatgagatgcctgctgacttgccttcattagctgctgattttgttgaaagtaaggatgtttgcaaaaactatgctgaggcaaaggatgtcttcctgggcatgtttttgtatgaatatgcaagaaggcatcctgattactctgtcgtgctgctgctgagacttgccaagacatatgaaaccactctagagaagtgctgtgccgctgcagatcctcatgaatgctatgccaaagtgttcgatgaatttaaacctcttgtggaagagcctcagaatttaatcaaacaaaattgtgagctttttgagcagcttggagagtacaaattccagaatgcgctattagttcgttacaccaagaaagtaccccaagtgtcaactccaactcttgtagaggtctcaagaaacctaggaaaagtgggcagcaaatgttgtaaacatcctgaagcaaaaagaatgccctgtgcagaagactatctatccgtggtcctgaaccagttatgtgtgttgcatgagaaaacgccagtaagtgacagagtcaccaaatgctgcacagaatccttggtgaacaggcgaccatgcttttcagctctggaagtcgatgaaacatacgttcccaaagagtttaatgctgaaacattcaccttccatgcagatatatgcacactttctgagaaggagagacaaatcaagaaacaaactgcacttgttgagctcgtgaaacacaagcccaaggcaacaaaagagcaactgaaagctgttatggatgatttcgcagcttttgtagagaagtgctgcaaggctgacgataaggagacctgctttgccgaggagggtaaaaaacttgttgctgcaagtcaagctgccttaggcttaggcagcggcggcggcggcagcggcggcggcggatctggtggaggtggcagtggaggagggggatccgcgcgcaacggggaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata 89 nucleic acidgatgcacacaagagtgaggttgctcatcggtttaaagatttgggagaagaaaatttcaaagccttgencodinggtgttgattgcctttgctcagtatcttcagcagtccccatttgaagatcatgtaaaattagtgaatgaagSEQ ID NO: 59taactgaatttgcaaaaacatgtgttgctgatgagtcagctgaaaattgtgacaaatcacttcataccattttggagacaaattatgcacagttgcaactatcgtgaaacctatggtgaaatggctgactgctgtgcaaaacaagaacctgagagaaatgaatgcttcttgcaacacaaagatgacaacccaaacctcccccgattggtgagaccagaggttgatgtgatgtgcactgcttttcatgacaatgaagagacatttttgaaaaaatacttatatgaaattgccagaagacatccttacttttatgccccggaactccttttctttgctaaaaggtataaagctgcttttacagaatgttgccaagctgctgataaagctgcctgcctgttgccaaagctcgatgaacttcgggatgaagggaaggcttcgtctgccaaacagagactcaagtgtgccagtctccaaaaatttggagaaagagctttcaaagcatgggcagtagctcgcctgagccagagatttcccaaagctgagtttgcagaagtttccaagttagtgacagatcttaccaaagtccacacggaatgctgccatggagatctgcttgaatgtgctgatgacagggcggaccttgccaagtatatctgtgaaaatcaagattcgatctccagtaaactgaaggaatgctgtgaaaaacctctgttggaaaaatcccactgcattgccgaagtggaaaatgatgagatgcctgctgacttgccttcattagctgctgattttgttgaaagtaaggatgtttgcaaaaactatgctgaggcaaaggatgtcttcctgggcatgtttttgtatgaatatgcaagaaggcatcctgattactctgtcgtgctgctgctgagacttgccaagacatatgaaaccactctagagaagtgctgtgccgctgcagatcctcatgaatgctatgccaaagtgttcgatgaatttaaacctcttgtggaagagcctcagaatttaatcaaacaaaattgtgagattttgagcagcttggagagtacaaattccagaatgcgctattagttcgttacaccaagaaagtaccccaagtgtcaactccaactcttgtagaggtctcaagaaacctaggaaaagtgggcagcaaatgttgtaaacatcctgaagcaaaaagaatgccctgtgcagaagactatctatccgtggtcctgaaccagttatgtgtgttgcatgagaaaacgccagtaagtgacagagtcaccaaatgctgcacagaatccttggtgaacaggcgaccatgcttttcagctctggaagtcgatgaaacatacgttcccaaagagtttaatgctgaaacattcaccttccatgcagatatatgcacactttctgagaaggagagacaaatcaagaaacaaactgcacttgttgagctcgtgaaacacaagcccaaggcaacaaaagagcaactgaaagctgttatggatgatttcgcagcttttgtagagaagtgctgcaaggctgacgataaggagacctgctttgccgaggagggtaaaaaacttgttgctgcaagtcaagctgccttaggcttagcacctgcccccgctccagcacccgccccagcccctgctcccgcaccagctcctgcaccagcgcgcaacggggaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata 90 nucleic acidgatgcacacaagagtgaggttgctcatcggtttaaagatttgggagaagaaaatttcaaagccttgencodinggtgttgattgcctttgctcagtatcttcagcagtccccatttgaagatcatgtaaaattagtgaatgaagSEQ ID NO: 60taactgaatttgcaaaaacatgtgttgctgatgagtcagctgaaaattgtgacaaatcacttcatacc(Codonctttttggagacaaattatgcacagttgcaactatcgtgaaacctatggtgaaatggctgactgctgtoptimization 1)gcaaaacaagaacctgagagaaatgaatgcttcttgcaacacaaagatgacaacccaaacctcccccgattggtgagaccagaggttgatgtgatgtgcactgcttttcatgacaatgaagagacatttttgaaaaaatacttatatgaaattgccagaagacatccttacttttatgccccggaactccttttctttgctaaaaggtataaagctgcttttacagaatgttgccaagctgctgataaagctgcctgcctgttgccaaagctcgatgaacttcgggatgaagggaaggcttcgtctgccaaacagagactcaagtgtgccagtctccaaaaatttggagaaagagctttcaaagcatgggcagtagctcgcctgagccagagatttcccaaagctgagtttgcagaagtttccaagttagtgacagatcttaccaaagtccacacggaatgctgccatggagatctgcttgaatgtgctgatgacagggcggaccttgccaagtatatctgtgaaaatcaagattcgatctccagtaaactgaaggaatgctgtgaaaaacctctgttggaaaaatcccactgcattgccgaagtggaaaatgatgagatgcctgctgacttgccttcattagctgctgattttgttgaaagtaaggatgtttgcaaaaactatgctgaggcaaaggatgtcttcctgggcatgtttttgtatgaatatgcaagaaggcatcctgattactctgtcgtgctgctgctgagacttgccaagacatatgaaaccactctagagaagtgctgtgccgctgcagatcctcatgaatgctatgccaaagtgttcgatgaatttaaacctcttgtggaagagcctcagaatttaatcaaacaaaattgtgagattttgagcagatggagagtacaaattccagaatgcgctattagttcgttacaccaagaaagtaccccaagtgtcaactccaactcttgtagaggtctcaagaaacctaggaaaagtgggcagcaaatgttgtaaacatcctgaagcaaaaagaatgccctgtgcagaagactatctatccgtggtcctgaaccagttatgtgtgttgcatgagaaaacgccagtaagtgacagagtcaccaaatgctgcacagaatccttggtgaacaggcgaccatgcttttcagctctggaagtcgatgaaacatacgttcccaaagagtttaatgctgaaacattcaccttccatgcagatatatgcacactttctgagaaggagagacaaatcaagaaacaaactgcacttgttgagctcgtgaaacacaagcccaaggcaacaaaagagcaactgaaagctgttatggatgatttcgcagcttttgtagagaagtgctgcaaggctgacgataaggagacctgctttgccgaggagggtaaaaaacttgttgctgcaagtcaagctgccttaggcttaggcagcggcggcggcggcagcggcggcggcggatctggtggaggtggcagtggaggagggggatccggcggcggcggcagcggcggcggcggatctggtggaggtggcagtggaggagggggatccgcgcgcaacggggaccactgtccgctcgggcccgggcgttgctgccgtctgcacacggtccgcgcgtcgctggaagacctgggctgggccgattgggtgctgtcgccacgggaggtgcaagtgaccatgtgcatcggcgcgtgcccgagccagttccgggcggcaaacatgcacgcgcagatcaagacgagcctgcaccgcctgaagcccgacacggtgccagcgccctgctgcgtgcccgccagctacaatcccatggtgctcattcaaaagaccgacaccggggtgtcgctccagacctatgatgacttgttagccaaagactgccactgcata 91 nucleic acidgacgcccacaagagcgaggtggcccaccggttcaaggacctgggcgaggagaacttcaaggc encodingcctggtgctgatcgccttcgcccagtacctgcagcagtccccatcgaggaccacgtgaagctggSEQ ID NO: 70tgaacgaggtgaccgagttcgccaagacctgcgtggccgacgagagcgccgagaactgcgacaagagcctgcacaccctgttcggcgacaagctgtgcaccgtggccaccctgcgggagacctacggcgagatggccgactgctgcgccaagcaggagcccgagcggaacgagtgatcctgcagcacaaggacgacaaccccaacctgccccggctggtgcggcccgaggtggacgtgatgtgcaccgccttccacgacaacgaggagaccttcctgaagaagtacctgtacgagatcgcccggcggcacccctacttctacgcccccgagctgctgttcttcgccaagcggtacaaggccgccttcaccgagtgctgccaggccgccgacaaggccgcctgcctgctgcccaagctggacgagctgcgggacgagggcaaggccagcagcgccaagcagcggctgaagtgcgccagcctgcagaagttcggcgagcgggccttcaaggcctgggccgtggcccggctgagccagcggttccccaaggccgagttcgccgaggtgagcaagctggtgaccgacctgaccaaggtgcacaccgagtgctgccacggcgacctgctggagtgcgccgacgaccgggccgacctggccaagtacatctgcgagaaccaggacagcatcagcagcaagctgaaggagtgctgcgagaagcccctgctggagaagagccactgcatcgccgaggtggagaacgacgagatgcccgccgacctgcccagcctggccgccgacttcgtggagagcaaggacgtgtgcaagaactacgccgaggccaaggacgtgttcctgggcatgttcctgtacgagtacgcccggcggcaccccgactacagcgtggtgctgctgctgcggctggccaagacctacgagaccaccctggagaagtgctgcgccgccgccgacccccacgagtgctacgccaaggtgttcgacgagttcaagcccctggtggaggagccccagaacctgatcaagcagaactgcgagctgttcgagcagctgggcgagtacaagttccagaacgccctgctggtgcggtacaccaagaaggtgccccaggtgagcacccccaccctggtggaggtgagccggaacctgggcaaggtgggcagcaagtgctgcaagcaccccgaggccaagcggatgccctgcgccgaggactacctgagcgtggtgctgaaccagctgtgcgtgctgcacgagaagacccccgtgagcgaccgggtgaccaagtgctgcaccgagagcctggtgaaccggcggccctgatcagcgccctggaggtggacgagacctacgtgcccaaggagttcaacgccgagaccttcaccttccacgccgacatctgcaccctgagcgagaaggagcggcagatcaagaagcagaccgccctggtggagctggtgaagcacaagcccaaggccaccaaggagcagctgaaggccgtgatggacgacttcgccgccttcgtggagaagtgctgcaaggccgacgacaaggagacctgcttcgccgaggagggcaagaagctggtggccgccagccaggccgccctgggcctgggcagcggcggcggcggcagcggcggcggcggatctggtggaggtggcagtggaggagggggatccgctcgcaacggtgaccactgccctctgggtcctggtcgctgctgccgcctgcacaccgttcgcgcttctctggaagacctgggttgggctgactgggttctgtctcctcgcgaagttcaggttaccatgtgcatcggtgcttgcccttctcagttccgcgctgctaacatgcacgcttggatcaaaacctctctgcaccgcctgaaacctgacaccgttcctgctccttgctgcgttcctgcttcttacaaccctatggttctgatccagaaaaccgacaccggtgtttctctgcagacctacgacgacctgctggctaaagactgccactgc atc 92HSA (C34S)- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV GS-(GGGGS)₈-TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC GDF15AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK (deletion 4)KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDD LLAKDCHCI 93 HSA (C34S)-DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV GS-(GGGGS)₈-TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC GDF15AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK (deletion 5)KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDD LLAKDCHCI 946x histidine tag- EFHHHHHHDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHSA (C34S)- HVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRET GS-(GGGGS)₈-YGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFH HRV3C siteDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADK GDF15AACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSGGGGSGGGGSGGGGSGGGGSLEVLFQGPARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLL AKDCHCI 95 nucleic acidGATGCACACAAGAGTGAGGTTGCTCATCGGTTTAAAGATTTGGG encodingAGAAGAAAATTTCAAAGCCTTGGTGTTGATTGCCTTTGCTCAGTA SEQ ID NO: 92TCTTCAGCAGTCCCCATTTGAAGATCATGTAAAATTAGTGAATGA (CodonAGTAACTGAATTTGCAAAAACATGTGTTGCTGATGAGTCAGCTG optimization 1)AAAATTGTGACAAATCACTTCATACCCTTTTTGGAGACAAATTATGCACAGTTGCAACTCTTCGTGAAACCTATGGTGAAATGGCTGACTGCTGTGCAAAACAAGAACCTGAGAGAAATGAATGCTTCTTGCAACACAAAGATGACAACCCAAACCTCCCCCGATTGGTGAGACCAGAGGTTGATGTGATGTGCACTGCTTTTCATGACAATGAAGAGACATTTTTGAAAAAATACTTATATGAAATTGCCAGAAGACATCCTTACTTTTATGCCCCGGAACTCCTTTTCTTTGCTAAAAGGTATAAAGCTGCTTTTACAGAATGTTGCCAAGCTGCTGATAAAGCTGCCTGCCTGTTGCCAAAGCTCGATGAACTTCGGGATGAAGGGAAGGCTTCGTCTGCCAAACAGAGACTCAAGTGTGCCAGTCTCCAAAAATTTGGAGAAAGAGCTTTCAAAGCATGGGCAGTAGCTCGCCTGAGCCAGAGATTTCCCAAAGCTGAGTTTGCAGAAGTTTCCAAGTTAGTGACAGATCTTACCAAAGTCCACACGGAATGCTGCCATGGAGATCTGCTTGAATGTGCTGATGACAGGGCGGACCTTGCCAAGTATATCTGTGAAAATCAAGATTCGATCTCCAGTAAACTGAAGGAATGCTGTGAAAAACCTCTGTTGGAAAAATCCCACTGCATTGCCGAAGTGGAAAATGATGAGATGCCTGCTGACTTGCCTTCATTAGCTGCTGATTTTGTTGAAAGTAAGGATGTTTGCAAAAACTATGCTGAGGCAAAGGATGTCTTCCTGGGCATGTTTTTGTATGAATATGCAAGAAGGCATCCTGATTACTCTGTCGTGCTGCTGCTGAGACTTGCCAAGACATATGAAACCACTCTAGAGAAGTGCTGTGCCGCTGCAGATCCTCATGAATGCTATGCCAAAGTGTTCGATGAATTTAAACCTCTTGTGGAAGAGCCTCAGAATTTAATCAAACAAAATTGTGAGCTTTTTGAGCAGCTTGGAGAGTACAAATTCCAGAATGCGCTATTAGTTCGTTACACCAAGAAAGTACCCCAAGTGTCAACTCCAACTCTTGTAGAGGTCTCAAGAAACCTAGGAAAAGTGGGCAGCAAATGTTGTAAACATCCTGAAGCAAAAAGAATGCCCTGTGCAGAAGACTATCTATCCGTGGTCCTGAACCAGTTATGTGTGTTGCATGAGAAAACGCCAGTAAGTGACAGAGTCACCAAATGCTGCACAGAATCCTTGGTGAACAGGCGACCATGCTTTTCAGCTCTGGAAGTCGATGAAACATACGTTCCCAAAGAGTTTAATGCTGAAACATTCACCTTCCATGCAGATATATGCACACTTTCTGAGAAGGAGAGACAAATCAAGAAACAAACTGCACTTGTTGAGCTCGTGAAACACAAGCCCAAGGCAACAAAAGAGCAACTGAAAGCTGTTATGGATGATTTCGCAGCTTTTGTAGAGAAGTGCTGCAAGGCTGACGATAAGGAGACCTGCTTTGCCGAGGAGGGTAAAAAACTTGTTGCTGCAAGTCAAGCTGCCTTAGGGCTTGGAAGCGGCGGAGGGGGGAGTGGCGGCGGTGGCTCCGGGGGGGGCGGATCCGGCGGAGGGGGCAGCGGGGGTGGAGGGAGTGGCGGGGGAGGATCAGGGGGAGGAGGATCAGGAGGGGGCGGAAGTGATCATTGCCCTCTCGGGCCCGGACGGTGTTGCCGCCTCCACACTGTGAGGGCTTCACTTGAAGACCTTGGATGGGCCGACTGGGTGCTGTCCCCAAGAGAGGTACAAGTCACAATGTGTATTGGCGCCTGCCCCAGCCAGTTTCGCGCCGCTAACATGCACGCCCAGATAAAAACCAGCCTGCACCGCCTGAAGCCCGACACGGTGCCAGCGCCCTGCTGCGTGCCCGCCAGCTACAATCCCATGGTGCTCATTCAAAAGACCGACACCGGGGTGTCGCTCCAGACCTATGATGACTTGTTAGCCAAAGACTGCCACTGCATA 96 nucleic acidGGGGACCACTGTCCGCTCGGGCCCGGGCGTTGCTGCCGTCTGCA encodingCACGGTCCGCGCGTCGCTGGAAGACCTGGGCTGGGCCGATTGGG SEQ ID NO: 7TGCTGTCGCCACGGGAGGTGCAAGTGACCATGTGCATCGGCGCGTGCCCGAGCCAGTTCCGGGCGGCAAACATGCACGCGCAGATCAAGACGAGCCTGCACCGCCTGAAGCCCGACACGGTGCCAGCGCCCTGCTGCGTGCCCGCCAGCTACAATCCCATGGTGCTCATTCAAAAGACCGACACCGGGGTGTCGCTCCAGACCTATGATGACTTGTTAGC CAAAGACTGCCACTGCATA 97nucleic acid GACCACTGTCCGCTCGGGCCCGGGCGTTGCTGCCGTCTGCACAC encodingGGTCCGCGCGTCGCTGGAAGACCTGGGCTGGGCCGATTGGGTGC SEQ ID NO: 8TGTCGCCACGGGAGGTGCAAGTGACCATGTGCATCGGCGCGTGC (CodonCCGAGCCAGTTCCGGGCGGCAAACATGCACGCGCAGATCAAGAC optimization 1)GAGCCTGCACCGCCTGAAGCCCGACACGGTGCCAGCGCCCTGCTGCGTGCCCGCCAGCTACAATCCCATGGTGCTCATTCAAAAGACCGACACCGGGGTGTCGCTCCAGACCTATGATGACTTGTTAGCCAA AGACTGCCACTGCATA 98nucleic acid GATCATTGCCCTCTCGGGCCCGGACGGTGTTGCCGCCTCCACACT encodingGTGAGGGCTTCACTTGAAGACCTTGGATGGGCCGACTGGGTGCT SEQ ID NO: 8GTCCCCAAGAGAGGTACAAGTCACAATGTGTATTGGCGCCTGCC (CodonCCAGCCAGTTTCGCGCCGCTAACATGCACGCCCAGATAAAAACC optimization 2)AGCCTGCACCGCCTGAAGCCCGACACGGTGCCAGCGCCCTGCTGCGTGCCCGCCAGCTACAATCCCATGGTGCTCATTCAAAAGACCGACACCGGGGTGTCGCTCCAGACCTATGATGACTTGTTAGCCAAA GACTGCCACTGCATA 99nucleic acid GATCATTGTCCCCTTGGACCGGGTAGATGCTGTCGCCTGCACACT encodingGTGCGGGCTTCACTGGAGGACCTCGGCTGGGCTGACTGGGTGCT SEQ ID NO: 8GTCCCCACGGGAGGTGCAAGTGACCATGTGCATCGGCGCCTGTC (CodonCTTCGCAATTCCGGGCCGCGAATATGCACGCCCAGATCAAGACC optimization 3)TCCCTGCATCGCCTCAAGCCCGACACTGTGCCTGCTCCATGCTGTGTGCCGGCCTCCTATAACCCCATGGTGCTGATCCAGAAAACCGATACCGGCGTCAGCCTGCAGACGTATGATGATCTGCTGGCCAAGG ACTGCCATTGCATC 100nucleic acid CACTGTCCGCTCGGGCCCGGGCGTTGCTGCCGTCTGCACACGGTC encodingCGCGCGTCGCTGGAAGACCTGGGCTGGGCCGATTGGGTGCTGTC SEQ ID NO: 9GCCACGGGAGGTGCAAGTGACCATGTGCATCGGCGCGTGCCCGA (CodonGCCAGTTCCGGGCGGCAAACATGCACGCGCAGATCAAGACGAG optimization 1)CCTGCACCGCCTGAAGCCCGACACGGTGCCAGCGCCCTGCTGCGTGCCCGCCAGCTACAATCCCATGGTGCTCATTCAAAAGACCGACACCGGGGTGTCGCTCCAGACCTATGATGACTTGTTAGCCAAAGA CTGCCACTGCATA 101nucleic acid CATTGCCCTCTCGGGCCCGGACGGTGTTGCCGCCTCCACACTGTG encodingAGGGCTTCACTTGAAGACCTTGGATGGGCCGACTGGGTGCTGTC SEQ ID NO: 9CCCAAGAGAGGTACAAGTCACAATGTGTATTGGCGCCTGCCCCA (CodonGCCAGTTTCGCGCCGCTAACATGCACGCCCAGATAAAAACCAGC optimization 2)CTGCACCGCCTGAAGCCCGACACGGTGCCAGCGCCCTGCTGCGTGCCCGCCAGCTACAATCCCATGGTGCTCATTCAAAAGACCGACACCGGGGTGTCGCTCCAGACCTATGATGACTTGTTAGCCAAAGAC TGCCACTGCATA 102nucleic acid CATTGTCCCCTTGGACCGGGTAGATGCTGTCGCCTGCACACTGTG encodingCGGGCTTCACTGGAGGACCTCGGCTGGGCTGACTGGGTGCTGTC SEQ ID NO: 9CCCACGGGAGGTGCAAGTGACCATGTGCATCGGCGCCTGTCCTT (CodonCGCAATTCCGGGCCGCGAATATGCACGCCCAGATCAAGACCTCC optimization 3)CTGCATCGCCTCAAGCCCGACACTGTGCCTGCTCCATGCTGTGTGCCGGCCTCCTATAACCCCATGGTGCTGATCCAGAAAACCGATACCGGCGTCAGCCTGCAGACGTATGATGATCTGCTGGCCAAGGACT GCCATTGCATC 103nucleic acid TGTCCGCTCGGGCCCGGGCGTTGCTGCCGTCTGCACACGGTCCGC encodingGCGTCGCTGGAAGACCTGGGCTGGGCCGATTGGGTGCTGTCGCC SEQ ID NO: 10ACGGGAGGTGCAAGTGACCATGTGCATCGGCGCGTGCCCGAGCC (CodonAGTTCCGGGCGGCAAACATGCACGCGCAGATCAAGACGAGCCTG optimization 1)CACCGCCTGAAGCCCGACACGGTGCCAGCGCCCTGCTGCGTGCCCGCCAGCTACAATCCCATGGTGCTCATTCAAAAGACCGACACCGGGGTGTCGCTCCAGACCTATGATGACTTGTTAGCCAAAGACTGC CACTGCATA 104 nucleic acidTGCCCTCTCGGGCCCGGACGGTGTTGCCGCCTCCACACTGTGAG encodingGGCTTCACTTGAAGACCTTGGATGGGCCGACTGGGTGCTGTCCC SEQ ID NO: 10CAAGAGAGGTACAAGTCACAATGTGTATTGGCGCCTGCCCCAGC (CodonCAGTTTCGCGCCGCTAACATGCACGCCCAGATAAAAACCAGCCT optimization 2)GCACCGCCTGAAGCCCGACACGGTGCCAGCGCCCTGCTGCGTGCCCGCCAGCTACAATCCCATGGTGCTCATTCAAAAGACCGACACCGGGGTGTCGCTCCAGACCTATGATGACTTGTTAGCCAAAGACTG CCACTGCATA 105 nucleic acidTGTCCCCTTGGACCGGGTAGATGCTGTCGCCTGCACACTGTGCGG encodingGCTTCACTGGAGGACCTCGGCTGGGCTGACTGGGTGCTGTCCCC SEQ ID NO: 10ACGGGAGGTGCAAGTGACCATGTGCATCGGCGCCTGTCCTTCGC (CodonAATTCCGGGCCGCGAATATGCACGCCCAGATCAAGACCTCCCTG optimization 3)CATCGCCTCAAGCCCGACACTGTGCCTGCTCCATGCTGTGTGCCGGCCTCCTATAACCCCATGGTGCTGATCCAGAAAACCGATACCGGCGTCAGCCTGCAGACGTATGATGATCTGCTGGCCAAGGACTGCC ATTGCATC 106 nucleic acidTGCCGTCTGCACACGGTCCGCGCGTCGCTGGAAGACCTGGGCTG encodingGGCCGATTGGGTGCTGTCGCCACGGGAGGTGCAAGTGACCATGT SEQ ID NO: 11GCATCGGCGCGTGCCCGAGCCAGTTCCGGGCGGCAAACATGCAC (CodonGCGCAGATCAAGACGAGCCTGCACCGCCTGAAGCCCGACACGGT optimization 1)GCCAGCGCCCTGCTGCGTGCCCGCCAGCTACAATCCCATGGTGCTCATTCAAAAGACCGACACCGGGGTGTCGCTCCAGACCTATGATGACTTGTTAGCCAAAGACTGCCACTGCATA 107 nucleic acidTGCCGCCTCCACACTGTGAGGGCTTCACTTGAAGACCTTGGATG encodingGGCCGACTGGGTGCTGTCCCCAAGAGAGGTACAAGTCACAATGT SEQ ID NO: 11GTATTGGCGCCTGCCCCAGCCAGTTTCGCGCCGCTAACATGCAC (CodonGCCCAGATAAAAACCAGCCTGCACCGCCTGAAGCCCGACACGGT optimization 2)GCCAGCGCCCTGCTGCGTGCCCGCCAGCTACAATCCCATGGTGCTCATTCAAAAGACCGACACCGGGGTGTCGCTCCAGACCTATGATGACTTGTTAGCCAAAGACTGCCACTGCATA 108 nucleic acidTGTCGCCTGCACACTGTGCGGGCTTCACTGGAGGACCTCGGCTG encodingGGCTGACTGGGTGCTGTCCCCACGGGAGGTGCAAGTGACCATGT SEQ ID NO: 11GCATCGGCGCCTGTCCTTCGCAATTCCGGGCCGCGAATATGCAC (CodonGCCCAGATCAAGACCTCCCTGCATCGCCTCAAGCCCGACACTGT optimization 3)GCCTGCTCCATGCTGTGTGCCGGCCTCCTATAACCCCATGGTGCTGATCCAGAAAACCGATACCGGCGTCAGCCTGCAGACGTATGATGATCTGCTGGCCAAGGACTGCCATTGCATC 109  nucleic acidGATGCGCACAAGTCGGAAGTGGCCCATCGCTTTAAGGACCTGGG encodingAGAAGAGAACTTCAAGGCCCTGGTCCTGATCGCGTTCGCCCAGT SEQ ID NO: 60ACCTCCAGCAGTCCCCGTTTGAGGACCACGTCAAGCTTGTGAAC (CodonGAAGTGACCGAGTTCGCAAAGACTTGTGTGGCCGATGAGTCCGC optimization 2)CGAAAACTGCGACAAGTCCCTGCACACCTTGTTCGGAGACAAGCTGTGCACCGTCGCGACTTTGCGGGAGACTTACGGCGAAATGGCGGACTGCTGCGCAAAGCAGGAGCCCGAAAGGAACGAGTGCTTCCTGCAACACAAGGACGACAACCCGAACCTTCCGAGACTCGTGCGGCCTGAGGTCGACGTGATGTGCACTGCATTCCATGATAACGAAGAAACATTCCTGAAGAAGTACCTGTATGAAATTGCCAGACGCCACCCGTACTTCTACGCCCCCGAACTGCTGTTCTTCGCCAAGAGATACAAGGCCGCCTTTACCGAATGTTGTCAAGCCGCCGATAAGGCAGCGTGCCTGCTGCCGAAGTTGGACGAGCTCAGGGACGAAGGAAAGGCCTCGTCCGCCAAGCAGAGGCTGAAGTGCGCGTCGCTCCAGAAGTTTGGAGAGCGGGCTTTTAAGGCCTGGGCAGTGGCTAGGTTGAGCCAGAGGTTCCCCAAGGCGGAGTTTGCCGAAGTGTCCAAGCTCGTGACTGACCTGACTAAAGTCCATACCGAATGCTGCCACGGCGATCTGCTCGAATGCGCAGATGACCGGGCGGATTTGGCCAAGTACATTTGCGAAAACCAAGACTCCATAAGCTCCAAGCTGAAGGAGTGCTGTGAAAAGCCTCTGCTCGAGAAGTCCCACTGTATCGCCGAGGTGGAGAACGACGAAATGCCGGCAGACCTCCCTAGCCTGGCAGCCGACTTCGTCGAATCCAAGGACGTGTGCAAGAACTACGCCGAAGCGAAGGACGTGTTCCTGGGAATGTTCCTGTACGAGTACGCCAGACGGCATCCAGACTACTCCGTGGTGCTTCTCTTGCGGCTGGCCAAGACTTATGAAACGACCCTGGAGAAATGTTGCGCTGCTGCTGACCCACACGAGTGCTACGCCAAAGTGTTCGACGAGTTTAAGCCTCTCGTGGAGGAACCCCAGAACCTCATCAAGCAGAACTGCGAACTTTTCGAGCAGCTCGGGGAGTACAAGTTCCAAAACGCGCTGCTTGTCCGCTACACCAAGAAAGTGCCGCAAGTGTCCACACCGACCCTCGTGGAAGTGTCCAGGAACCTGGGCAAAGTCGGAAGCAAATGTTGCAAGCACCCCGAAGCCAAGCGCATGCCGTGCGCAGAGGACTACCTTTCGGTGGTGTTGAACCAGCTCTGCGTCCTGCACGAAAAGACCCCGGTGTCAGACCGCGTGACCAAGTGCTGTACCGAAAGCCTCGTGAATCGGCGCCCCTGCTTCTCGGCCCTGGAGGTGGACGAAACTTACGTGCCGAAAGAGTTCAACGCGGAAACCTTCACCTTTCATGCCGATATCTGCACCCTGTCCGAGAAGGAGCGGCAGATCAAGAAGCAGACCGCCCTGGTGGAGCTTGTGAAACACAAGCCGAAGGCCACTAAGGAACAGCTGAAGGCCGTCATGGACGATTTCGCTGCCTTCGTCGAGAAGTGCTGCAAGGCCGACGACAAGGAGACTTGCTTCGCTGAAGAAGGGAAGAAGCTTGTGGCCGCTAGCCAGGCTGCACTGGGACTGGGTAGCGGTGGAGGGGGATCAGGGGGTGGTGGATCGGGAGGAGGAGGATCAGGAGGTGGCGGCTCAGGAGGAGGCGGATCAGGCGGTGGAGGATCCGGAGGCGGAGGATCGGGTGGAGGAGGCTCAGCGAGGAACGGGGATCATTGTCCCCTTGGACCGGGTAGATGCTGTCGCCTGCACACTGTGCGGGCTTCACTGGAGGACCTCGGCTGGGCTGACTGGGTGCTGTCCCCACGGGAGGTGCAAGTGACCATGTGCATCGGCGCCTGTCCTTCGCAATTCCGGGCCGCGAATATGCACGCCCAGATCAAGACCTCCCTGCATCGCCTCAAGCCCGACACTGTGCCTGCTCCATGCTGTGTGCCGGCCTCCTATAACCCCATGGTGCTGATCCAGAAAACCGATACCGGCGTCAGCCTGCAGACGTATGATGATCTGCTGGC CAAGGACTGCCATTGCATC 110nucleic acid GATGCGCACAAGTCGGAAGTGGCCCATCGCTTTAAGGACCTGGG encodingAGAAGAGAACTTCAAGGCCCTGGTCCTGATCGCGTTCGCCCAGT SEQ ID NO: 92ACCTCCAGCAGTCCCCGTTTGAGGACCACGTCAAGCTTGTGAAC (CodonGAAGTGACCGAGTTCGCAAAGACTTGTGTGGCCGATGAGTCCGC optimization 2)CGAAAACTGCGACAAGTCCCTGCACACCTTGTTCGGAGACAAGCTGTGCACCGTCGCGACTTTGCGGGAGACTTACGGCGAAATGGCGGACTGCTGCGCAAAGCAGGAGCCCGAAAGGAACGAGTGCTTCCTGCAACACAAGGACGACAACCCGAACCTTCCGAGACTCGTGCGGCCTGAGGTCGACGTGATGTGCACTGCATTCCATGATAACGAAGAAACATTCCTGAAGAAGTACCTGTATGAAATTGCCAGACGCCACCCGTACTTCTACGCCCCCGAACTGCTGTTCTTCGCCAAGAGATACAAGGCCGCCTTTACCGAATGTTGTCAAGCCGCCGATAAGGCAGCGTGCCTGCTGCCGAAGTTGGACGAGCTCAGGGACGAAGGAAAGGCCTCGTCCGCCAAGCAGAGGCTGAAGTGCGCGTCGCTCCAGAAGTTTGGAGAGCGGGCTTTTAAGGCCTGGGCAGTGGCTAGGTTGAGCCAGAGGTTCCCCAAGGCGGAGTTTGCCGAAGTGTCCAAGCTCGTGACTGACCTGACTAAAGTCCATACCGAATGCTGCCACGGCGATCTGCTCGAATGCGCAGATGACCGGGCGGATTTGGCCAAGTACATTTGCGAAAACCAAGACTCCATAAGCTCCAAGCTGAAGGAGTGCTGTGAAAAGCCTCTGCTCGAGAAGTCCCACTGTATCGCCGAGGTGGAGAACGACGAAATGCCGGCAGACCTCCCTAGCCTGGCAGCCGACTTCGTCGAATCCAAGGACGTGTGCAAGAACTACGCCGAAGCGAAGGACGTGTTCCTGGGAATGTTCCTGTACGAGTACGCCAGACGGCATCCAGACTACTCCGTGGTGCTTCTCTTGCGGCTGGCCAAGACTTATGAAACGACCCTGGAGAAATGTTGCGCTGCTGCTGACCCACACGAGTGCTACGCCAAAGTGTTCGACGAGTTTAAGCCTCTCGTGGAGGAACCCCAGAACCTCATCAAGCAGAACTGCGAACTTTTCGAGCAGCTCGGGGAGTACAAGTTCCAAAACGCGCTGCTTGTCCGCTACACCAAGAAAGTGCCGCAAGTGTCCACACCGACCCTCGTGGAAGTGTCCAGGAACCTGGGCAAAGTCGGAAGCAAATGTTGCAAGCACCCCGAAGCCAAGCGCATGCCGTGCGCAGAGGACTACCTTTCGGTGGTGTTGAACCAGCTCTGCGTCCTGCACGAAAAGACCCCGGTGTCAGACCGCGTGACCAAGTGCTGTACCGAAAGCCTCGTGAATCGGCGCCCCTGCTTCTCGGCCCTGGAGGTGGACGAAACTTACGTGCCGAAAGAGTTCAACGCGGAAACCTTCACCTTTCATGCCGATATCTGCACCCTGTCCGAGAAGGAGCGGCAGATCAAGAAGCAGACCGCCCTGGTGGAGCTTGTGAAACACAAGCCGAAGGCCACTAAGGAACAGCTGAAGGCCGTCATGGACGATTTCGCTGCCTTCGTCGAGAAGTGCTGCAAGGCCGACGACAAGGAGACTTGCTTCGCTGAAGAAGGGAAGAAGCTTGTGGCCGCTAGCCAGGCTGCACTGGGACTGGGTAGCGGTGGAGGGGGATCAGGGGGTGGTGGATCGGGAGGAGGAGGATCAGGAGGTGGCGGCTCAGGAGGAGGCGGATCAGGCGGTGGAGGATCCGGAGGCGGAGGATCGGGTGGAGGAGGCTCAGATCATTGTCCCCTTGGACCGGGTAGATGCTGTCGCCTGCACACTGTGCGGGCTTCACTGGAGGACCTCGGCTGGGCTGACTGGGTGCTGTCCCCACGGGAGGTGCAAGTGACCATGTGCATCGGCGCCTGTCCTTCGCAATTCCGGGCCGCGAATATGCACGCCCAGATCAAGACCTCCCTGCATCGCCTCAAGCCCGACACTGTGCCTGCTCCATGCTGTGTGCCGGCCTCCTATAACCCCATGGTGCTGATCCAGAAAACCGATACCGGCGTCAGCCTGCAGACGTATGATGATCTGCTGGCCAAGGACTGCC ATTGCATC 111 HSA (C34S)-GS-DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV (GGGGS)₈-TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC (Deletion 5)AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK GDF15KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDD LLAKDCHCI 112HSA (C34S)-GS- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV (GGGGS)₈-TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC (deletion 14)AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK GDF15KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI 113 (Deletion 2)HKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTE HSA (C34S)-GS-FAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAK (GGGGS)₈-QEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYL (deletion 4)YEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDEL GDF15RDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAK DCHCI 114 DNA for SEQCACAAGAGTGAGGTTGCTCATCGGTTTAAAGATTTGGGAGAAGA ID NO: 113AAATTTCAAAGCCTTGGTGTTGATTGCCTTTGCTCAGTATCTTCA (Deletion 2)GCAGTCCCCATTTGAAGATCATGTAAAATTAGTGAATGAAGTAA HSA (C34S)-GS-CTGAATTTGCAAAAACATGTGTTGCTGATGAGTCAGCTGAAAAT (GGGGS)₈-TGTGACAAATCACTTCATACCCTTTTTGGAGACAAATTATGCACA (deletion 4)GTTGCAACTCTTCGTGAAACCTATGGTGAAATGGCTGACTGCTGT GDF15GCAAAACAAGAACCTGAGAGAAATGAATGCTTCTTGCAACACAAAGATGACAACCCAAACCTCCCCCGATTGGTGAGACCAGAGGTTGATGTGATGTGCACTGCTTTTCATGACAATGAAGAGACATTTTTGAAAAAATACTTATATGAAATTGCCAGAAGACATCCTTACTTTTATGCCCCGGAACTCCTTTTCTTTGCTAAAAGGTATAAAGCTGCTTTTACAGAATGTTGCCAAGCTGCTGATAAAGCTGCCTGCCTGTTGCCAAAGCTCGATGAACTTCGGGATGAAGGGAAGGCTTCGTCTGCCAAACAGAGACTCAAGTGTGCCAGTCTCCAAAAATTTGGAGAAAGAGCTTTCAAAGCATGGGCAGTAGCTCGCCTGAGCCAGAGATTTCCCAAAGCTGAGTTTGCAGAAGTTTCCAAGTTAGTGACAGATCTTACCAAAGTCCACACGGAATGCTGCCATGGAGATCTGCTTGAATGTGCTGATGACAGGGCGGACCTTGCCAAGTATATCTGTGAAAATCAAGATTCGATCTCCAGTAAACTGAAGGAATGCTGTGAAAAACCTCTGTTGGAAAAATCCCACTGCATTGCCGAAGTGGAAAATGATGAGATGCCTGCTGACTTGCCTTCATTAGCTGCTGATTTTGTTGAAAGTAAGGATGTTTGCAAAAACTATGCTGAGGCAAAGGATGTCTTCCTGGGCATGTTTTTGTATGAATATGCAAGAAGGCATCCTGATTACTCTGTCGTGCTGCTGCTGAGACTTGCCAAGACATATGAAACCACTCTAGAGAAGTGCTGTGCCGCTGCAGATCCTCATGAATGCTATGCCAAAGTGTTCGATGAATTTAAACCTCTTGTGGAAGAGCCTCAGAATTTAATCAAACAAAATTGTGAGCTTTTTGAGCAGCTTGGAGAGTACAAATTCCAGAATGCGCTATTAGTTCGTTACACCAAGAAAGTACCCCAAGTGTCAACTCCAACTCTTGTAGAGGTCTCAAGAAACCTAGGAAAAGTGGGCAGCAAATGTTGTAAACATCCTGAAGCAAAAAGAATGCCCTGTGCAGAAGACTATCTATCCGTGGTCCTGAACCAGTTATGTGTGTTGCATGAGAAAACGCCAGTAAGTGACAGAGTCACCAAATGCTGCACAGAATCCTTGGTGAACAGGCGACCATGCTTTTCAGCTCTGGAAGTCGATGAAACATACGTTCCCAAAGAGTTTAATGCTGAAACATTCACCTTCCATGCAGATATATGCACACTTTCTGAGAAGGAGAGACAAATCAAGAAACAAACTGCACTTGTTGAGCTCGTGAAACACAAGCCCAAGGCAACAAAAGAGCAACTGAAAGCTGTTATGGATGATTTCGCAGCTTTTGTAGAGAAGTGCTGCAAGGCTGACGATAAGGAGACCTGCTTTGCCGAGGAGGGTAAAAAACTTGTTGCTGCAAGTCAAGCTGCCTTAGGGCTTGGAAGCGGCGGAGGGGGGAGTGGCGGCGGTGGCTCCGGGGGGGGCGGATCCGGCGGAGGGGGCAGCGGGGGTGGAGGGAGTGGCGGGGGAGGATCAGGGGGAGGAGGATCAGGAGGGGGCGGAAGTGATCATTGCCCTCTCGGGCCCGGACGGTGTTGCCGCCTCCACACTGTGAGGGCTTCACTTGAAGACCTTGGATGGGCCGACTGGGTGCTGTCCCCAAGAGAGGTACAAGTCACAATGTGTATTGGCGCCTGCCCCAGCCAGTTTCGCGCCGCTAACATGCACGCCCAGATAAAAACCAGCCTGCACCGCCTGAAGCCCGACACGGTGCCAGCGCCCTGCTGCGTGCCCGCCAGCTACAATCCCATGGTGCTCATTCAAAAGACCGACACCGGGGTGTCGCTCCAGACCTATGATGACTTGTTAGCCAAAGACTGCCACTGCATA 115 HSA (C34S)-DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV GA-(GGGGA)₈-TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC (deletion 4)AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK GDF15KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGAGGGGADHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTY DDLLAKDCHCI 116DNA for SEQ GATGCACACAAGAGTGAGGTTGCTCATCGGTTTAAAGATTTGGG ID NO: 115AGAAGAAAATTTCAAAGCCTTGGTGTTGATTGCCTTTGCTCAGTA HSA (C34S)-TCTTCAGCAGTCCCCATTTGAAGATCATGTAAAATTAGTGAATGA GA-(GGGGA)₈-AGTAACTGAATTTGCAAAAACATGTGTTGCTGATGAGTCAGCTG (deletion 4)AAAATTGTGACAAATCACTTCATACCCTTTTTGGAGACAAATTAT GDF15GCACAGTTGCAACTCTTCGTGAAACCTATGGTGAAATGGCTGACTGCTGTGCAAAACAAGAACCTGAGAGAAATGAATGCTTCTTGCAACACAAAGATGACAACCCAAACCTCCCCCGATTGGTGAGACCAGAGGTTGATGTGATGTGCACTGCTTTTCATGACAATGAAGAGACATTTTTGAAAAAATACTTATATGAAATTGCCAGAAGACATCCTTACTTTTATGCCCCGGAACTCCTTTTCTTTGCTAAAAGGTATAAAGCTGCTTTTACAGAATGTTGCCAAGCTGCTGATAAAGCTGCCTGCCTGTTGCCAAAGCTCGATGAACTTCGGGATGAAGGGAAGGCTTCGTCTGCCAAACAGAGACTCAAGTGTGCCAGTCTCCAAAAATTTGGAGAAAGAGCTTTCAAAGCATGGGCAGTAGCTCGCCTGAGCCAGAGATTTCCCAAAGCTGAGTTTGCAGAAGTTTCCAAGTTAGTGACAGATCTTACCAAAGTCCACACGGAATGCTGCCATGGAGATCTGCTTGAATGTGCTGATGACAGGGCGGACCTTGCCAAGTATATCTGTGAAAATCAAGATTCGATCTCCAGTAAACTGAAGGAATGCTGTGAAAAACCTCTGTTGGAAAAATCCCACTGCATTGCCGAAGTGGAAAATGATGAGATGCCTGCTGACTTGCCTTCATTAGCTGCTGATTTTGTTGAAAGTAAGGATGTTTGCAAAAACTATGCTGAGGCAAAGGATGTCTTCCTGGGCATGTTTTTGTATGAATATGCAAGAAGGCATCCTGATTACTCTGTCGTGCTGCTGCTGAGACTTGCCAAGACATATGAAACCACTCTAGAGAAGTGCTGTGCCGCTGCAGATCCTCATGAATGCTATGCCAAAGTGTTCGATGAATTTAAACCTCTTGTGGAAGAGCCTCAGAATTTAATCAAACAAAATTGTGAGCTTTTTGAGCAGCTTGGAGAGTACAAATTCCAGAATGCGCTATTAGTTCGTTACACCAAGAAAGTACCCCAAGTGTCAACTCCAACTCTTGTAGAGGTCTCAAGAAACCTAGGAAAAGTGGGCAGCAAATGTTGTAAACATCCTGAAGCAAAAAGAATGCCCTGTGCAGAAGACTATCTATCCGTGGTCCTGAACCAGTTATGTGTGTTGCATGAGAAAACGCCAGTAAGTGACAGAGTCACCAAATGCTGCACAGAATCCTTGGTGAACAGGCGACCATGCTTTTCAGCTCTGGAAGTCGATGAAACATACGTTCCCAAAGAGTTTAATGCTGAAACATTCACCTTCCATGCAGATATATGCACACTTTCTGAGAAGGAGAGACAAATCAAGAAACAAACTGCACTTGTTGAGCTCGTGAAACACAAGCCCAAGGCAACAAAAGAGCAACTGAAAGCTGTTATGGATGATTTCGCAGCTTTTGTAGAGAAGTGCTGCAAGGCTGACGATAAGGAGACCTGCTTTGCCGAGGAGGGTAAAAAACTTGTTGCTGCAAGTCAAGCTGCCTTAGGGCTTGGTGCTGGAGGAGGCGGGGCGGGCGGCGGGGGTGCCGGTGGGGGTGGCGCAGGGGGAGGTGGTGCGGGTGGTGGTGGGGCTGGTGGGGGAGGTGCAGGCGGTGGCGGTGCCGGGGGGGGTGGCGCGGATCATTGCCCTCTCGGGCCCGGACGGTGTTGCCGCCTCCACACTGTGAGGGCTTCACTTGAAGACCTTGGATGGGCCGACTGGGTGCTGTCCCCAAGAGAGGTACAAGTCACAATGTGTATTGGCGCCTGCCCCAGCCAGTTTCGCGCCGCTAACATGCACGCCCAGATAAAAACCAGCCTGCACCGCCTGAAGCCCGACACGGTGCCAGCGCCCTGCTGCGTGCCCGCCAGCTACAATCCCATGGTGCTCATTCAAAAGACCGACACCGGGGTGTCGCTCCAGACCTATGATGACTTGTTAGCCAAAGACTGCCACTGCATA 117 HSA (C34S)-DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV (AP)₁₀-TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC (deletion 4)AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK GDF15KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLAPAPAPAPAPAPAPAPAPAPDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI 118 DNA for SEQGATGCACACAAGAGTGAGGTTGCTCATCGGTTTAAAGATTTGGG ID NO: 117AGAAGAAAATTTCAAAGCCTTGGTGTTGATTGCCTTTGCTCAGTA HSA (C34S)-TCTTCAGCAGTCCCCATTTGAAGATCATGTAAAATTAGTGAATGA (AP)₁₀-AGTAACTGAATTTGCAAAAACATGTGTTGCTGATGAGTCAGCTG (deletion 4)AAAATTGTGACAAATCACTTCATACCCTTTTTGGAGACAAATTAT GDF15GCACAGTTGCAACTCTTCGTGAAACCTATGGTGAAATGGCTGACTGCTGTGCAAAACAAGAACCTGAGAGAAATGAATGCTTCTTGCAACACAAAGATGACAACCCAAACCTCCCCCGATTGGTGAGACCAGAGGTTGATGTGATGTGCACTGCTTTTCATGACAATGAAGAGACATTTTTGAAAAAATACTTATATGAAATTGCCAGAAGACATCCTTACTTTTATGCCCCGGAACTCCTTTTCTTTGCTAAAAGGTATAAAGCTGCTTTTACAGAATGTTGCCAAGCTGCTGATAAAGCTGCCTGCCTGTTGCCAAAGCTCGATGAACTTCGGGATGAAGGGAAGGCTTCGTCTGCCAAACAGAGACTCAAGTGTGCCAGTCTCCAAAAATTTGGAGAAAGAGCTTTCAAAGCATGGGCAGTAGCTCGCCTGAGCCAGAGATTTCCCAAAGCTGAGTTTGCAGAAGTTTCCAAGTTAGTGACAGATCTTACCAAAGTCCACACGGAATGCTGCCATGGAGATCTGCTTGAATGTGCTGATGACAGGGCGGACCTTGCCAAGTATATCTGTGAAAATCAAGATTCGATCTCCAGTAAACTGAAGGAATGCTGTGAAAAACCTCTGTTGGAAAAATCCCACTGCATTGCCGAAGTGGAAAATGATGAGATGCCTGCTGACTTGCCTTCATTAGCTGCTGATTTTGTTGAAAGTAAGGATGTTTGCAAAAACTATGCTGAGGCAAAGGATGTCTTCCTGGGCATGTTTTTGTATGAATATGCAAGAAGGCATCCTGATTACTCTGTCGTGCTGCTGCTGAGACTTGCCAAGACATATGAAACCACTCTAGAGAAGTGCTGTGCCGCTGCAGATCCTCATGAATGCTATGCCAAAGTGTTCGATGAATTTAAACCTCTTGTGGAAGAGCCTCAGAATTTAATCAAACAAAATTGTGAGCTTTTTGAGCAGCTTGGAGAGTACAAATTCCAGAATGCGCTATTAGTTCGTTACACCAAGAAAGTACCCCAAGTGTCAACTCCAACTCTTGTAGAGGTCTCAAGAAACCTAGGAAAAGTGGGCAGCAAATGTTGTAAACATCCTGAAGCAAAAAGAATGCCCTGTGCAGAAGACTATCTATCCGTGGTCCTGAACCAGTTATGTGTGTTGCATGAGAAAACGCCAGTAAGTGACAGAGTCACCAAATGCTGCACAGAATCCTTGGTGAACAGGCGACCATGCTTTTCAGCTCTGGAAGTCGATGAAACATACGTTCCCAAAGAGTTTAATGCTGAAACATTCACCTTCCATGCAGATATATGCACACTTTCTGAGAAGGAGAGACAAATCAAGAAACAAACTGCACTTGTTGAGCTCGTGAAACACAAGCCCAAGGCAACAAAAGAGCAACTGAAAGCTGTTATGGATGATTTCGCAGCTTTTGTAGAGAAGTGCTGCAAGGCTGACGATAAGGAGACCTGCTTTGCCGAGGAGGGTAAAAAACTTGTTGCTGCAAGTCAAGCTGCCTTAGGGCTTGCACCAGCCCCTGCCCCTGCACCTGCACCTGCTCCCGCACCGGCTCCAGCCCCAGCTCCGGATCATTGCCCTCTCGGGCCCGGACGGTGTTGCCGCCTCCACACTGTGAGGGCTTCACTTGAAGACCTTGGATGGGCCGACTGGGTGCTGTCCCCAAGAGAGGTACAAGTCACAATGTGTATTGGCGCCTGCCCCAGCCAGTTTCGCGCCGCTAACATGCACGCCCAGATAAAAACCAGCCTGCACCGCCTGAAGCCCGACACGGTGCCAGCGCCCTGCTGCGTGCCCGCCAGCTACAATCCCATGGTGCTCATTCAAAAGACCGACACCGGGGTGTCGCTCCAGACCTATGATGACTTGTTAGCCAAA GACTGCCACTGCATA 119HSA (C34S)- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV (AP)₁₂-TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC (deletion 4)AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK GDF15KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLAPAPAPAPAPAPAPAPAPAPAPAPDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI 120 DNA for SEQGATGCACACAAGAGTGAGGTTGCTCATCGGTTTAAAGATTTGGG ID NO: 119AGAAGAAAATTTCAAAGCCTTGGTGTTGATTGCCTTTGCTCAGTA HSA (C34S)-TCTTCAGCAGTCCCCATTTGAAGATCATGTAAAATTAGTGAATGA (AP)₁₂-AGTAACTGAATTTGCAAAAACATGTGTTGCTGATGAGTCAGCTG (deletion 4)AAAATTGTGACAAATCACTTCATACCCTTTTTGGAGACAAATTAT GDF15GCACAGTTGCAACTCTTCGTGAAACCTATGGTGAAATGGCTGACTGCTGTGCAAAACAAGAACCTGAGAGAAATGAATGCTTCTTGCAACACAAAGATGACAACCCAAACCTCCCCCGATTGGTGAGACCAGAGGTTGATGTGATGTGCACTGCTTTTCATGACAATGAAGAGACATTTTTGAAAAAATACTTATATGAAATTGCCAGAAGACATCCTTACTTTTATGCCCCGGAACTCCTTTTCTTTGCTAAAAGGTATAAAGCTGCTTTTACAGAATGTTGCCAAGCTGCTGATAAAGCTGCCTGCCTGTTGCCAAAGCTCGATGAACTTCGGGATGAAGGGAAGGCTTCGTCTGCCAAACAGAGACTCAAGTGTGCCAGTCTCCAAAAATTTGGAGAAAGAGCTTTCAAAGCATGGGCAGTAGCTCGCCTGAGCCAGAGATTTCCCAAAGCTGAGTTTGCAGAAGTTTCCAAGTTAGTGACAGATCTTACCAAAGTCCACACGGAATGCTGCCATGGAGATCTGCTTGAATGTGCTGATGACAGGGCGGACCTTGCCAAGTATATCTGTGAAAATCAAGATTCGATCTCCAGTAAACTGAAGGAATGCTGTGAAAAACCTCTGTTGGAAAAATCCCACTGCATTGCCGAAGTGGAAAATGATGAGATGCCTGCTGACTTGCCTTCATTAGCTGCTGATTTTGTTGAAAGTAAGGATGTTTGCAAAAACTATGCTGAGGCAAAGGATGTCTTCCTGGGCATGTTTTTGTATGAATATGCAAGAAGGCATCCTGATTACTCTGTCGTGCTGCTGCTGAGACTTGCCAAGACATATGAAACCACTCTAGAGAAGTGCTGTGCCGCTGCAGATCCTCATGAATGCTATGCCAAAGTGTTCGATGAATTTAAACCTCTTGTGGAAGAGCCTCAGAATTTAATCAAACAAAATTGTGAGCTTTTTGAGCAGCTTGGAGAGTACAAATTCCAGAATGCGCTATTAGTTCGTTACACCAAGAAAGTACCCCAAGTGTCAACTCCAACTCTTGTAGAGGTCTCAAGAAACCTAGGAAAAGTGGGCAGCAAATGTTGTAAACATCCTGAAGCAAAAAGAATGCCCTGTGCAGAAGACTATCTATCCGTGGTCCTGAACCAGTTATGTGTGTTGCATGAGAAAACGCCAGTAAGTGACAGAGTCACCAAATGCTGCACAGAATCCTTGGTGAACAGGCGACCATGCTTTTCAGCTCTGGAAGTCGATGAAACATACGTTCCCAAAGAGTTTAATGCTGAAACATTCACCTTCCATGCAGATATATGCACACTTTCTGAGAAGGAGAGACAAATCAAGAAACAAACTGCACTTGTTGAGCTCGTGAAACACAAGCCCAAGGCAACAAAAGAGCAACTGAAAGCTGTTATGGATGATTTCGCAGCTTTTGTAGAGAAGTGCTGCAAGGCTGACGATAAGGAGACCTGCTTTGCCGAGGAGGGTAAAAAACTTGTTGCTGCAAGTCAAGCTGCCTTAGGGCTTGCACCAGCCCCTGCCCCTGCACCTGCACCTGCTCCCGCACCGGCTCCAGCCCCAGCTCCGGCTCCAGCTCCTGATCATTGCCCTCTCGGGCCCGGACGGTGTTGCCGCCTCCACACTGTGAGGGCTTCACTTGAAGACCTTGGATGGGCCGACTGGGTGCTGTCCCCAAGAGAGGTACAAGTCACAATGTGTATTGGCGCCTGCCCCAGCCAGTTTCGCGCCGCTAACATGCACGCCCAGATAAAAACCAGCCTGCACCGCCTGAAGCCCGACACGGTGCCAGCGCCCTGCTGCGTGCCCGCCAGCTACAATCCCATGGTGCTCATTCAAAAGACCGACACCGGGGTGTCGCTCCAGACCTATGATGACTT GTTAGCCAAAGACTGCCACTGCATA121 HSA (C34S)- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV GGS-TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC (EGKSSGSGSESKST)₃-AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK GGS-KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKL (deletion4)DELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAE GDF15FAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGSEGKSSGSGSESKSTEGKSSGSGSESKSTEGKSSGSGSESKSTGGSDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYD DLLAKDCHCI 122DNA for SEQ GATGCACACAAGAGTGAGGTTGCTCATCGGTTTAAAGATTTGGG ID NO: 121AGAAGAAAATTTCAAAGCCTTGGTGTTGATTGCCTTTGCTCAGTA HSA (C34S)-TCTTCAGCAGTCCCCATTTGAAGATCATGTAAAATTAGTGAATGA GGS-AGTAACTGAATTTGCAAAAACATGTGTTGCTGATGAGTCAGCTG (EGKSSGSGSESKST)₃-AAAATTGTGACAAATCACTTCATACCCTTTTTGGAGACAAATTAT GGS-GCACAGTTGCAACTCTTCGTGAAACCTATGGTGAAATGGCTGAC (deletion4)TGCTGTGCAAAACAAGAACCTGAGAGAAATGAATGCTTCTTGCA GDF15ACACAAAGATGACAACCCAAACCTCCCCCGATTGGTGAGACCAGAGGTTGATGTGATGTGCACTGCTTTTCATGACAATGAAGAGACATTTTTGAAAAAATACTTATATGAAATTGCCAGAAGACATCCTTACTTTTATGCCCCGGAACTCCTTTTCTTTGCTAAAAGGTATAAAGCTGCTTTTACAGAATGTTGCCAAGCTGCTGATAAAGCTGCCTGCCTGTTGCCAAAGCTCGATGAACTTCGGGATGAAGGGAAGGCTTCGTCTGCCAAACAGAGACTCAAGTGTGCCAGTCTCCAAAAATTTGGAGAAAGAGCTTTCAAAGCATGGGCAGTAGCTCGCCTGAGCCAGAGATTTCCCAAAGCTGAGTTTGCAGAAGTTTCCAAGTTAGTGACAGATCTTACCAAAGTCCACACGGAATGCTGCCATGGAGATCTGCTTGAATGTGCTGATGACAGGGCGGACCTTGCCAAGTATATCTGTGAAAATCAAGATTCGATCTCCAGTAAACTGAAGGAATGCTGTGAAAAACCTCTGTTGGAAAAATCCCACTGCATTGCCGAAGTGGAAAATGATGAGATGCCTGCTGACTTGCCTTCATTAGCTGCTGATTTTGTTGAAAGTAAGGATGTTTGCAAAAACTATGCTGAGGCAAAGGATGTCTTCCTGGGCATGTTTTTGTATGAATATGCAAGAAGGCATCCTGATTACTCTGTCGTGCTGCTGCTGAGACTTGCCAAGACATATGAAACCACTCTAGAGAAGTGCTGTGCCGCTGCAGATCCTCATGAATGCTATGCCAAAGTGTTCGATGAATTTAAACCTCTTGTGGAAGAGCCTCAGAATTTAATCAAACAAAATTGTGAGCTTTTTGAGCAGCTTGGAGAGTACAAATTCCAGAATGCGCTATTAGTTCGTTACACCAAGAAAGTACCCCAAGTGTCAACTCCAACTCTTGTAGAGGTCTCAAGAAACCTAGGAAAAGTGGGCAGCAAATGTTGTAAACATCCTGAAGCAAAAAGAATGCCCTGTGCAGAAGACTATCTATCCGTGGTCCTGAACCAGTTATGTGTGTTGCATGAGAAAACGCCAGTAAGTGACAGAGTCACAAAATGCTGTACTGAGAGCTTGGTCAACAGGCGGCCGTGCTTCAGCGCCCTCGAGGTGGATGAGACTTATGTCCCAAAGGAGTTTAATGCGGAAACTTTTACTTTCCACGCAGACATTTGCACCTTGTCTGAAAAGGAAAGACAGATTAAGAAACAGACTGCTCTTGTGGAACTGGTAAAACATAAACCAAAAGCTACGAAGGAGCAGCTTAAGGCTGTTATGGATGATTTCGCCGCGTTTGTCGAGAAGTGCTGCAAAGCGGACGATAAGGAAACTTGCTTTGCAGAGGAAGGTAAGAAACTCGTAGCGGCAAGTCAGGCTGCGCTTGGCCTTGGAGGCAGTGAAGGCAAATCCTCTGGGAGTGGCTCTGAAAGTAAATCCACCGAGGGCAAATCCAGTGGATCTGGGTCTGAATCTAAGTCTACCGAGGGGAAGTCTTCTGGCAGTGGGTCAGAATCTAAATCTACAGGCGGCTCTGACCATTGCCCGTTGGGACCAGGACGCTGCTGTCGCCTTCATACAGTGCGAGCGAGTTTGGAAGACCTGGGCTGGGCTGACTGGGTGCTTAGCCCTCGGGAGGTCCAGGTCACAATGTGCATTGGCGCGTGTCCCAGTCAATTTAGAGCAGCAAATATGCACGCCCAAATAAAAACCTCCCTGCATAGGCTTAAGCCAGATACTGTCCCCGCACCATGCTGTGTGCCTGCTTCTTACAATCCTATGGTACTCATCCAGAAGACCGACACGGGAGTTAGCCTCCAGACTTATGACGACCTCTTGGCTAAAGATT GCCATTGTATT 123HSA (C34S)-GS- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV (PGGGS)₈-TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC (deletion4)AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK GDF15KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSPGGGSPGGGSPGGGSPGGGSPGGGSPGGGSPGGGSPGGGSDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLL AKDCHCI 124 DNA for SEQGATGCACACAAGAGTGAGGTTGCTCATCGGTTTAAAGATTTGGG ID NO: 123AGAAGAAAATTTCAAAGCCTTGGTGTTGATTGCCTTTGCTCAGTA HSA (C34S)-GS-TCTTCAGCAGTCCCCATTTGAAGATCATGTAAAATTAGTGAATGA (PGGGS)₈-AGTAACTGAATTTGCAAAAACATGTGTTGCTGATGAGTCAGCTG (deletion4)AAAATTGTGACAAATCACTTCATACCCTTTTTGGAGACAAATTAT GDF15GCACAGTTGCAACTCTTCGTGAAACCTATGGTGAAATGGCTGACTGCTGTGCAAAACAAGAACCTGAGAGAAATGAATGCTTCTTGCAACACAAAGATGACAACCCAAACCTCCCCCGATTGGTGAGACCAGAGGTTGATGTGATGTGCACTGCTTTTCATGACAATGAAGAGACATTTTTGAAAAAATACTTATATGAAATTGCCAGAAGACATCCTTACTTTTATGCCCCGGAACTCCTTTTCTTTGCTAAAAGGTATAAAGCTGCTTTTACAGAATGTTGCCAAGCTGCTGATAAAGCTGCCTGCCTGTTGCCAAAGCTCGATGAACTTCGGGATGAAGGGAAGGCTTCGTCTGCCAAACAGAGACTCAAGTGTGCCAGTCTCCAAAAATTTGGAGAAAGAGCTTTCAAAGCATGGGCAGTAGCTCGCCTGAGCCAGAGATTTCCCAAAGCTGAGTTTGCAGAAGTTTCCAAGTTAGTGACAGATCTTACCAAAGTCCACACGGAATGCTGCCATGGAGATCTGCTTGAATGTGCTGATGACAGGGCGGACCTTGCCAAGTATATCTGTGAAAATCAAGATTCGATCTCCAGTAAACTGAAGGAATGCTGTGAAAAACCTCTGTTGGAAAAATCCCACTGCATTGCCGAAGTGGAAAATGATGAGATGCCTGCTGACTTGCCTTCATTAGCTGCTGATTTTGTTGAAAGTAAGGATGTTTGCAAAAACTATGCTGAGGCAAAGGATGTCTTCCTGGGCATGTTTTTGTATGAATATGCAAGAAGGCATCCTGATTACTCTGTCGTGCTGCTGCTGAGACTTGCCAAGACATATGAAACCACTCTAGAGAAGTGCTGTGCCGCTGCAGATCCTCATGAATGCTATGCCAAAGTGTTCGATGAATTTAAACCTCTTGTGGAAGAGCCTCAGAATTTAATCAAACAAAATTGTGAGCTTTTTGAGCAGCTTGGAGAGTACAAATTCCAGAATGCGCTATTAGTTCGTTACACCAAGAAAGTACCCCAAGTGTCAACTCCAACTCTTGTAGAGGTCTCAAGAAACCTAGGAAAAGTGGGCAGCAAATGTTGTAAACATCCTGAAGCAAAAAGAATGCCCTGTGCAGAAGACTATCTATCCGTGGTCCTGAACCAGTTATGTGTGTTGCATGAGAAAACGCCAGTAAGTGACAGAGTCACGAAATGTTGCACAGAGTCACTGGTCAACAGGAGACCTTGCTTCTCCGCTCTTGAGGTTGACGAAACGTATGTCCCAAAAGAGTTCAACGCCGAAACGTTTACGTTTCATGCGGACATATGCACTCTCAGTGAGAAGGAGCGACAAATCAAAAAACAGACTGCTCTTGTAGAGTTGGTAAAACACAAACCTAAAGCAACAAAAGAGCAATTGAAAGCTGTGATGGACGATTTTGCAGCTTTCGTAGAAAAGTGCTGCAAGGCCGACGATAAAGAAACCTGTTTCGCTGAAGAAGGCAAAAAACTTGTTGCGGCATCTCAGGCCGCTCTTGGACTTGGGAGCCCGGGTGGCGGGTCTCCAGGCGGAGGCTCTCCGGGCGGAGGTAGTCCCGGAGGGGGTAGTCCGGGCGGCGGTTCTCCAGGTGGAGGTTCTCCTGGTGGTGGCAGTCCTGGCGGAGGATCTGATCACTGTCCCCTTGGGCCCGGGAGGTGCTGCCGACTTCATACAGTTCGCGCCAGCCTTGAAGATTTGGGGTGGGCCGACTGGGTGTTGAGCCCGAGAGAGGTCCAAGTCACGATGTGTATTGGAGCCTGTCCCTCTCAATTCCGAGCCGCAAATATGCATGCGCAAATAAAGACGAGTCTCCATCGGTTGAAGCCTGATACTGTCCCAGCTCCGTGCTGCGTCCCCGCGAGTTATAATCCCATGGTCCTTATACAGAAAACAGACACTGGTGTCAGCCTTCAGACGTATGACGATTTGCTTGCTAAAGACTGTCATTGTATT 125 HSA (C34S)-GS-DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV (AGGGS)₈-TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC (deletion 4)AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK GDF15KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGSAGGGSAGGGSAGGGSAGGGSAGGGSAGGGSAGGGSAGGGSDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDD LLAKDCHCI 126 DNA for SEQGATGCACACAAGAGTGAGGTTGCTCATCGGTTTAAAGATTTGGG ID NO: 125AGAAGAAAATTTCAAAGCCTTGGTGTTGATTGCCTTTGCTCAGTATCTTCAGCAGTCCCCATTTGAAGATCATGTAAAATTAGTGAATGAAGTAACTGAATTTGCAAAAACATGTGTTGCTGATGAGTCAGCTGAAAATTGTGACAAATCACTTCATACCCTTTTTGGAGACAAATTATGCACAGTTGCAACTCTTCGTGAAACCTATGGTGAAATGGCTGACTGCTGTGCAAAACAAGAACCTGAGAGAAATGAATGCTTCTTGCAACACAAAGATGACAACCCAAACCTCCCCCGATTGGTGAGACCAGAGGTTGATGTGATGTGCACTGCTTTTCATGACAATGAAGAGACATTTTTGAAAAAATACTTATATGAAATTGCCAGAAGACATCCTTACTTTTATGCCCCGGAACTCCTTTTCTTTGCTAAAAGGTATAAAGCTGCTTTTACAGAATGTTGCCAAGCTGCTGATAAAGCTGCCTGCCTGTTGCCAAAGCTCGATGAACTTCGGGATGAAGGGAAGGCTTCGTCTGCCAAACAGAGACTCAAGTGTGCCAGTCTCCAAAAATTTGGAGAAAGAGCTTTCAAAGCATGGGCAGTAGCTCGCCTGAGCCAGAGATTTCCCAAAGCTGAGTTTGCAGAAGTTTCCAAGTTAGTGACAGATCTTACCAAAGTCCACACGGAATGCTGCCATGGAGATCTGCTTGAATGTGCTGATGACAGGGCGGACCTTGCCAAGTATATCTGTGAAAATCAAGATTCGATCTCCAGTAAACTGAAGGAATGCTGTGAAAAACCTCTGTTGGAAAAATCCCACTGCATTGCCGAAGTGGAAAATGATGAGATGCCTGCTGACTTGCCTTCATTAGCTGCTGATTTTGTTGAAAGTAAGGATGTTTGCAAAAACTATGCTGAGGCAAAGGATGTCTTCCTGGGCATGTTTTTGTATGAATATGCAAGAAGGCATCCTGATTACTCTGTCGTGCTGCTGCTGAGACTTGCCAAGACATATGAAACCACTCTAGAGAAGTGCTGTGCCGCTGCAGATCCTCATGAATGCTATGCCAAAGTGTTCGATGAATTTAAACCTCTTGTGGAAGAGCCTCAGAATTTAATCAAACAAAATTGTGAGCTTTTTGAGCAGCTTGGAGAGTACAAATTCCAGAATGCGCTATTAGTTCGTTACACCAAGAAAGTACCCCAAGTGTCAACTCCAACTCTTGTAGAGGTCTCAAGAAACCTAGGAAAAGTGGGCAGCAAATGTTGTAAACATCCTGAAGCAAAAAGAATGCCCTGTGCAGAAGACTATCTATCCGTGGTCCTGAACCAGTTATGTGTGTTGCATGAGAAAACGCCAGTAAGTGACAGAGTCACTAAATGTTGTACCGAGTCTCTTGTTAATAGGCGGCCATGCTTCAGTGCATTGGAAGTCGACGAAACCTATGTACCAAAGGAGTTCAACGCAGAAACATTTACATTCCATGCTGATATCTGCACATTGAGCGAGAAAGAGAGACAGATTAAGAAACAGACAGCGCTTGTTGAACTGGTTAAACACAAACCAAAAGCTACCAAGGAGCAGCTTAAGGCAGTAATGGATGACTTCGCGGCCTTTGTCGAGAAATGTTGTAAAGCGGATGATAAAGAGACATGCTTCGCCGAAGAGGGCAAAAAACTTGTAGCGGCAAGCCAGGCCGCACTGGGTCTCGGTAGTGCGGGCGGTGGTTCAGCGGGGGGAGGATCTGCAGGTGGTGGCTCAGCGGGTGGCGGTAGCGCTGGGGGGGGCTCCGCAGGTGGGGGATCAGCAGGCGGCGGATCAGCCGGCGGTGGATCCGACCACTGTCCTCTCGGGCCTGGTCGGTGTTGCCGCCTCCATACTGTGCGCGCGTCTCTTGAGGATCTGGGGTGGGCTGATTGGGTTCTCTCTCCCCGCGAAGTGCAGGTGACCATGTGTATTGGTGCTTGCCCAAGTCAATTCCGAGCAGCTAACATGCACGCCCAGATCAAGACTAGCCTGCATCGGCTTAAGCCCGACACTGTTCCTGCCCCTTGCTGTGTTCCTGCATCTTATAATCCAATGGTCCTGATCCAGAAAACCGATACGGGTGTATCATTGCAAACATACGACGACTTGCTTGCCAAAGATTGCCATTGCATT 127 HSA (C34S)-DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEV GGS-TEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC (EGKSSGSGSESKST)₂-AKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK GGS-KYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKL (deletion4)DELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAE GDF15FAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGSEGKSSGSGSESKSTEGKSSGSGSESKSTGGSDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI 128 DNA for SEQGATGCACACAAGAGTGAGGTTGCTCATCGGTTTAAAGATTTGGG ID NO: 127AGAAGAAAATTTCAAAGCCTTGGTGTTGATTGCCTTTGCTCAGTATCTTCAGCAGTCCCCATTTGAAGATCATGTAAAATTAGTGAATGAAGTAACTGAATTTGCAAAAACATGTGTTGCTGATGAGTCAGCTGAAAATTGTGACAAATCACTTCATACCCTTTTTGGAGACAAATTATGCACAGTTGCAACTCTTCGTGAAACCTATGGTGAAATGGCTGACTGCTGTGCAAAACAAGAACCTGAGAGAAATGAATGCTTCTTGCAACACAAAGATGACAACCCAAACCTCCCCCGATTGGTGAGACCAGAGGTTGATGTGATGTGCACTGCTTTTCATGACAATGAAGAGACATTTTTGAAAAAATACTTATATGAAATTGCCAGAAGACATCCTTACTTTTATGCCCCGGAACTCCTTTTCTTTGCTAAAAGGTATAAAGCTGCTTTTACAGAATGTTGCCAAGCTGCTGATAAAGCTGCCTGCCTGTTGCCAAAGCTCGATGAACTTCGGGATGAAGGGAAGGCTTCGTCTGCCAAACAGAGACTCAAGTGTGCCAGTCTCCAAAAATTTGGAGAAAGAGCTTTCAAAGCATGGGCAGTAGCTCGCCTGAGCCAGAGATTTCCCAAAGCTGAGTTTGCAGAAGTTTCCAAGTTAGTGACAGATCTTACCAAAGTCCACACGGAATGCTGCCATGGAGATCTGCTTGAATGTGCTGATGACAGGGCGGACCTTGCCAAGTATATCTGTGAAAATCAAGATTCGATCTCCAGTAAACTGAAGGAATGCTGTGAAAAACCTCTGTTGGAAAAATCCCACTGCATTGCCGAAGTGGAAAATGATGAGATGCCTGCTGACTTGCCTTCATTAGCTGCTGATTTTGTTGAAAGTAAGGATGTTTGCAAAAACTATGCTGAGGCAAAGGATGTCTTCCTGGGCATGTTTTTGTATGAATATGCAAGAAGGCATCCTGATTACTCTGTCGTGCTGCTGCTGAGACTTGCCAAGACATATGAAACCACTCTAGAGAAGTGCTGTGCCGCTGCAGATCCTCATGAATGCTATGCCAAAGTGTTCGATGAATTTAAACCTCTTGTGGAAGAGCCTCAGAATTTAATCAAACAAAATTGTGAGCTTTTTGAGCAGCTTGGAGAGTACAAATTCCAGAATGCGCTATTAGTTCGTTACACCAAGAAAGTACCCCAAGTGTCAACTCCAACTCTTGTAGAGGTCTCAAGAAACCTAGGAAAAGTGGGCAGCAAATGTTGTAAACATCCTGAAGCAAAAAGAATGCCCTGTGCAGAAGACTATCTATCCGTGGTCCTGAACCAGTTATGTGTGTTGCATGAGAAAACGCCAGTAAGTGACAGAGTCACGAAATGCTGTACAGAATCCCTCGTGAATAGAAGGCCCTGCTTCTCTGCCCTTGAGGTGGACGAGACTTACGTCCCTAAGGAGTTTAACGCCGAGACCTTTACTTTTCATGCTGATATTTGCACCCTTTCCGAAAAGGAGCGGCAGATCAAGAAACAAACAGCCTTGGTGGAACTCGTAAAACATAAACCCAAAGCCACCAAGGAACAACTTAAAGCTGTTATGGATGACTTCGCAGCCTTCGTCGAGAAATGTTGCAAGGCGGATGATAAGGAAACGTGTTTTGCTGAGGAAGGGAAGAAGTTGGTTGCTGCCTCTCAAGCGGCTCTGGGGCTTGGCGGATCAGAGGGGAAGTCCTCCGGGTCCGGTAGCGAGTCCAAATCTACGGAAGGGAAGTCATCCGGTTCTGGGTCAGAGTCCAAATCCACAGGAGGATCAGACCATTGCCCATTGGGACCAGGACGATGTTGTCGCCTGCATACGGTAAGAGCGTCTCTGGAGGATCTCGGCTGGGCCGATTGGGTTCTCTCACCACGAGAAGTACAGGTCACAATGTGCATAGGAGCTTGTCCGAGCCAATTCCGGGCGGCTAATATGCACGCACAGATCAAGACCTCTTTGCACCGCTTGAAGCCCGATACCGTGCCAGCACCGTGTTGCGTCCCAGCATCTTACAACCCTATGGTTTTGATACAGAAAACTGACACAGGTGTGAGCCTCCAGACATATGATGATTTGCTGGCTAAGG ATTGCCACTGTATA

We claim:
 1. A fusion protein comprising: a. a half-life extensionprotein, b. a linker, and c. a GDF15 protein; wherein the fusion proteinis arranged from N-terminus to C-terminus in the order (a)-(b)-(c). 2.The fusion protein of claim 1, wherein the GDF15 protein comprises anamino acid sequence having at least 90% identity to SEQ ID NO: 6, 7, 8,9, 10 or
 11. 3. The fusion protein of claim 1, wherein the half-lifeextension protein is human serum albumin (HSA) or a functional variantthereof.
 4. The fusion protein of claim 3, wherein the half-lifeextension protein comprises an amino acid sequence having at least 90%identity to SEQ ID NO:
 1. 5. The fusion protein of claim 1, wherein thelinker comprises the sequence (GGGGS)n, wherein n is 2 to
 20. 6. Thefusion protein of claim 1, wherein the linker comprises the sequence(AP)n or (EAAAK)n, wherein n is 2 to
 20. 7. The fusion protein of claim1, wherein the linker comprises the amino acid sequence selected fromthe group consisting of (GGGGA)n, (PGGGS)n, (AGGGS)n orGGS-(EGKSSGSGSESKST)n-GGS wherein n is 2 to
 20. 8. A fusion proteincomprising an amino acid sequence having at least 90% sequence identityto SEQ ID NOs: 5, 25-30, 36-37, 40, 48, 55-56, 59-60, 64-75, 92, 113,115, 117, 119, 121, 123, 125, or
 127. 9. An isolated nucleic acidmolecule comprising a nucleotide sequence encoding the fusion protein ofclaim
 1. 10. A vector comprising a nucleic acid molecule encoding thefusion protein of claim
 1. 11. A method of producing the fusion proteinof claim 1, comprising: (1) culturing a host cell comprising a nucleicacid molecule encoding the fusion protein under a condition that thefusion protein is produced; and (2) recovering the fusion proteinproduced by the host cell.
 12. A pharmaceutical composition comprisingthe fusion protein of claim 1 and a pharmaceutically acceptable carrier.13. A method of treating or preventing a metabolic disorder, comprisingadministering to a subject in need thereof an effective amount of thepharmaceutical composition of claim
 12. 14. The method of claim 13wherein the metabolic disorder is selected from the group consisting ofa metabolic disorder selected from the group consisting of type 2diabetes, elevated glucose levels, elevated insulin levels, obesity,dyslipidemia, diabetic nephropathy; myocardial ischemic injury,congestive heart failure, and rheumatoid arthritis in a subject in needthereof, comprising administering to the subject a therapeuticallyeffective amount of a pharmaceutical composition comprising a fusionprotein comprising the amino acid sequence of SEQ ID NO: 60 or NO: 92,and a pharmaceutically acceptable carrier.
 15. A dimer comprising twopolypeptide chains, wherein each chain comprises from N-terminus toC-terminus an HSA region, a linker, and a GDF15 region; wherein the HSAregion comprises an ammo acid sequence having at least 90% identity toSEQ ID NOs: 1, 2, or 3, and wherein the GDF15 region comprises an aminoacid sequence having at least 90% identity to SEQ ID NOs: 6, 7, 8, 9,10, or
 11. 16. The dimer of claim 15, wherein the two polypeptide chainsare linked by disulfide bonds.
 17. The dimer of claim 15, wherein theHSA region and the GDF15 region are joined by a polypeptide linker. 18.The dimer of claim 15, wherein the polypeptide linker comprises thesequence (GGGGS)n, wherein n is 2 to
 20. 19. The dimer of claim 15,wherein the polypeptide linker comprises the sequence (AP)n or (EAAAK)n,wherein n is 2 to
 20. 20. The dimer of claims 17, wherein the linkercomprises the amino acid sequence selected from the group consisting of(GGGGA)n, (PGGGS)n, (AGGGS)n or GGS-(EGKSSGSGSESKST)n-GGS wherein n is 2to 20.